Adeno-associated virus purification methods

ABSTRACT

Provided herein are methods of producing an adeno-associated virus (AAV) product, methods of purifying adeno-associated virus, and methods of purifying full AAV capsids from a concentrated AAV fraction comprising empty AAV capsids and full AAV capsids.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/417,775 filed Nov. 4, 2016, which is hereby incorporated byreference in its entirety.

TECHNICAL FIELD

The invention relates to materials and methods of purifyingadeno-associated virus (AAV).

BACKGROUND

Adeno-associated virus (AAV) is a small, non-enveloped virus thatpackages a linear single-stranded DNA genome. AAV belongs to the familyParvoviridae and the genus Dependovirus, since productive infection byAAV occurs only in the presence of a helper virus, such as, for example,adenovirus or herpes virus. Even in the absence of a helper virus, AAV(serotype 2) can achieve latency by integrating into chromosome 19q13.4of a host human genome. It is the only mammalian DNA virus known to becapable of site-specific integration (Daya and Berns, ClinicalMicrobiology Reviews, pages 583-593 (2008)).

For AAV to be safely used in the clinic, AAV has been geneticallymodified at several locations within its genome. For example, the Repgene, which is required for viral replication, and the element requiredfor site-specific integration have been eliminated from the AAV genomein many viral vectors. These recombinant AAV (rAAV), exists in anextrachromosomal state and have very low integration efficiency into thegenomic DNA. The possibility of rAAV inducing random mutagenesis in ahost cell is thus reduced, if not eliminated altogether. Because ofthese properties and the lack of pathogenicity, rAAV has shown greatpromise as a gene therapy vector in multiple aspects of pre-clinical andclinical applications. New serotypes and self-complementary vectors arebeing tested in the clinic. Alongside these ongoing vector developments,continued effort has focused on scalable manufacturing processes thatcan efficiently generate high titer quantities of rAAV vectors with highpurity and potency.

Though the effort to design efficient, large-scale methods to purify anAAV product suitable for human administration has been great, therestill remains a need for better AAV purification methods. For example,current methods of generating AAV in cell culture result in theformation of “empty” capsids which have been shown to lead toT-cell-mediated immune responses against capsid antigen, leading tolow-grade hepatotoxicity and partial loss of expression (Wright, MolecTherapy 22(1): 1-2 (2014)). AAV purification methods which include stepsfor removing empty AAV capsids from the final AAV product are thereforedesired.

SUMMARY

A feature of AAV vector generation in cell culture is the formation ofan excess of “empty” capsids, which lack the vector genome. Such emptycapsids are unable to provide a therapeutic benefit associated withtransgene production. The effect of the empty capsids on clinicaloutcome is not clear. However, there is a potential for increasinginnate or adaptive immune responses to the vector, which then rendersempty capsids a concern in gene therapy contexts. Wright, MolecularTherapy 22: 1-2 (2014).

Provided herein are methods of producing an adeno-associated virus (AAV)product, methods of purifying AAV, and methods of purifying full AAVcapsids from a concentrated AAV fraction comprising empty AAV capsidsand full AAV capsids. The methods of the present disclosure areadvantageous over those known in the art, as the methods provided hereare suitable for large-scale production of AAV and provide a highlypure, potent product suitable for clinical use. In exemplary aspects,the methods described herein provide an AAV product comprising AAVparticles of a homogenous population and high purity. In exemplaryaspects, the methods described herein provide an AAV product comprisingfull-length vector DNA. In exemplary embodiments, the methods describedherein provide an AAV product that is substantially free of unwantedcontaminants, including but not limited to, empty AAV particles(including containing truncated or incomplete vector DNA), AAV particleswith incomplete protein composition and oligomerized structures, orcontaminating viruses, e.g., non AAV, lipid enveloped viruses. Inexemplary embodiments, the methods described herein provide an AAVproduct containing a high amount of DNA (cDNA) encoding of the proteinof interest.

In exemplary embodiments, the methods of the present disclosure comprisean ultracentrifugation step to separate full AAV capsids from empty AAVcapsids. In exemplary aspects, the methods comprise (i) loading into arotor a concentrated AAV fraction with at least two sugar solutions,each of which has a different sugar concentration, (ii) operating anultracentrifuge comprising the loaded rotor in batch mode to form asugar gradient, and (iii) obtaining a fraction of the sugar gradient toobtain an AAV fraction comprising full AAV capsids. In exemplaryaspects, the rotor is a zonal rotor.

In exemplary aspects, the at least two sugar solutions each comprises asugar at a concentration equivalent to a sucrose concentration rangingfrom about 45% (w/w) to about 65% (w/w) sucrose. In exemplary aspects,one sugar solution comprises a sugar at a concentration equivalent to asucrose concentration ranging from about 52% (w/w) to about 58% (w/w)sucrose and another sugar solution comprises a sugar at a concentrationequivalent to a sucrose concentration ranging from about 57% (w/w) toabout 63% (w/w) sucrose. Optionally, there is another sugar solutioncomprising a sugar at a concentration equivalent to a sucroseconcentration ranging from about 47% (w/w) to about 53% (w/w) sucrose.When loading to the bottom of the rotor or compartment, the order ofloading sequence can be first the sugar solution comprising a sugar at aconcentration equivalent to a sucrose concentration ranging from about47% (w/w) to about 53% (w/w) sucrose, if present, then the sugarsolution comprising a sugar at a concentration equivalent to a sucroseconcentration ranging from about 52% (w/w) to about 58% (w/w) sucrose,and then the sugar solution comprising a sugar at a concentrationequivalent to a sucrose concentration ranging from about 57% (w/w) toabout 63% (w/w) sucrose.

In certain embodiments, the sample to be processed is added prior to theaddition of the sugar solutions. In certain embodiments,ultracentrifugation is conducted in a continuous flow manner and thesample is loaded after a density gradient is achieved duringcentrifugation.

In certain embodiments, the methods of the present disclosure comprisean ultracentrifugation step to separate full AAV capsids from empty AAVcapsids. In exemplary aspects, the methods comprise (i) loading into arotor a concentrated AAV fraction with at least two sugar solutions,each of which has a different sugar concentration and each of whichcomprises a sugar at a concentration equivalent to a sucroseconcentration ranging from about 45% (w/w) to about 65% (w/w) sucrose,(ii) operating an ultracentrifuge comprising the loaded rotor in batchmode to form a sugar gradient, and (iii) obtaining a fraction of thesugar gradient to obtain an AAV fraction wherein at least or about 60%of the AAV particles in the AAV fraction are full AAV capsids. Inexemplary aspects, the rotor is a zonal rotor. In certain embodiments,the volume of the sugar solutions is greater than or equal to about 50%of the volume of the zonal rotor. In certain embodiments, the totalvolume of the sugar solutions and the AAV fraction is less than or equalto the volume of the zonal rotor. In certain embodiments, the ratio ofthe volume of the sugar solutions to the volume of the AAV fraction isless than or equal to one.

In certain embodiments, each sugar solution comprises a sugar at aconcentration equivalent to a sucrose concentration ranging from about50% (w/w) to about 60% (w/w) sucrose. In certain embodiments, each sugarsolution comprises a sugar at a concentration equivalent to a sucroseconcentration ranging from about 55% (w/w) to about 60% (w/w) sucrose.In certain embodiments, at least one of the sugar solutions comprisessugar at a concentration equivalent to a sucrose concentration greaterthan about 50% (w/w) sucrose. In certain embodiments, at least one ofthe sugar solutions comprises sugar at a concentration equivalent to asucrose concentration greater than about 55% (w/w) sucrose. In certainembodiments, at least one of the sugar solutions comprises sugar at aconcentration equivalent to a sucrose concentration ranging from about60% (w/w) to about 65% (w/w) sucrose.

In certain embodiments, one sugar solution comprises a sugar at aconcentration equivalent to a sucrose concentration ranging from about52% (w/w) to about 58% (w/w) sucrose, wherein a second sugar solutioncomprises a sugar at a concentration equivalent to a sucroseconcentration ranging from about 57% (w/w) to about 63% (w/w) sucrose,and optionally wherein a third sugar solution comprises a sugar at aconcentration equivalent to a sucrose concentration ranging from about47% (w/w) to about 53% (w/w) sucrose.

In certain aspects, two sugar solutions are loaded into the zonal rotor.In certain embodiments, two sugar solutions are loaded into the zonalrotor and wherein one sugar solution comprises a sugar at aconcentration equivalent to a sucrose concentration ranging from about52% (w/w) to about 58% (w/w) sucrose and a second sugar solutioncomprises a sugar at a concentration equivalent to a sucroseconcentration ranging from about 57% (w/w) to about 63% (w/w) sucrose.

In certain aspects, at least three sugar solutions are loaded into thezonal rotor. In certain embodiments, three sugar solutions are loadedinto the zonal rotor. In certain embodiments, three sugar solutions areloaded into the zonal rotor and wherein one sugar solution comprises asugar at a concentration equivalent to a sucrose concentration rangingfrom about 47% (w/w) to about 53% (w/w) sucrose, a second sugar solutioncomprises a sugar at a concentration equivalent to a sucroseconcentration ranging from about 52% (w/w) to about 58% (w/w) sucrose,and a third sugar solution comprises a sugar at a concentrationequivalent to a sucrose concentration ranging from about 57% (w/w) toabout 63% (w/w) sucrose.

In certain embodiments, the concentrated AAV fraction is loaded before asugar solution comprising a sugar at a concentration equivalent to asucrose concentration ranging from about 52% (w/w) to about 58% (w/w)sucrose, and wherein the sugar solution comprising a sugar at aconcentration equivalent to a sucrose concentration ranging from about52% (w/w) to about 58% (w/w) sucrose is loaded before a sugar solutioncomprising a sugar at a concentration equivalent to a sucroseconcentration ranging from about 57% (w/w) to about 63% (w/w) sucrose.

In certain embodiments, the concentrated AAV fraction is loaded before asugar solution comprising a sugar at a concentration equivalent to asucrose concentration ranging from about 47% (w/w) to about 53% (w/w)sucrose, wherein the sugar solution comprising a sugar at aconcentration equivalent to a sucrose concentration ranging from about47% (w/w) to about 53% (w/w) sucrose is loaded before a sugar solutioncomprising a sugar at a concentration equivalent to a sucroseconcentration ranging from about 52% (w/w) to about 58% (w/w) sucrose,and wherein the sugar solution comprising a sugar at a concentrationequivalent to a sucrose concentration ranging from about 52% (w/w) toabout 58% (w/w) sucrose is loaded before a sugar solution comprising asugar at a concentration equivalent to a sucrose concentration rangingfrom about 57% (w/w) to about 63% (w/w) sucrose.

In certain embodiments, each sugar solution is loaded in the zonal rotorat equal volumes. In certain embodiments, the sugar solution with thesmallest sugar concentration is loaded in the zonal rotor at a volumewhich is twice the volume of at least one of the other sugar solutionsin the zonal rotor. In certain embodiments, wherein the sugar solutionwith the smallest sugar concentration is loaded in the zonal rotor at avolume which is at least the volume of all other sugar solutionscombined in the zonal rotor. In certain embodiments, the sugar solutionwith the smallest sugar concentration is loaded in the zonal rotor at avolume which is at least twice the volume of the sugar solution with thelargest sugar concentration, optionally, wherein the volume of the sugarsolution with the largest sugar concentration is equal to the volume ofthe sugar solution with the intermediate sugar concentration. In certainembodiments, at least two sugar solutions are loaded into the zonalrotor, wherein the sugar solution with the smallest sugar concentrationis loaded in the zonal rotor at a volume which is at least twice thevolume of at least one other sugar solution in the zonal rotor. Incertain embodiments, at least two sugar solutions are loaded into thezonal rotor, wherein the sugar solution with the smallest sugarconcentration is loaded in the zonal rotor at a volume which is the samevolume of at least one other sugar solution in the zonal rotor. Incertain embodiments, the sugar solution with the smallest sugarconcentration is loaded in the zonal rotor at a volume which is half thevolume of the concentrated AAV fraction. In certain embodiments, wherein the ratio of the volume of the total sugar gradient to the volume ofthe AAV fraction loaded in the zonal rotor is from about 1:1 to about1:5.

In certain aspects, the AAV fraction comprises a buffered solution. Incertain embodiments, the buffered solution includes, without limitation,phosphate buffers, histidine (e.g., L-histidine), sodium citrate, HEPES,Tris, Bicine, glycine, N-glycylglycine, sodium acetate, sodiumcarbonate, glycyl glycine, lysine, arginine, sodium phosphate, andmixtures thereof. In certain embodiments, the AAV fraction comprisesTrisHCl and NaCl. In certain embodiments, the TrisHCl is at aconcentration of about 20 to about 50 mM and the NaCl is at aconcentration of about 150 mM to about 900 mM. In certain embodiments,the NaCl is at a concentration of about 500 mM to about 750 mM. Incertain embodiments, the buffered solution has a pH of about 7.4 toabout 9.0. In certain embodiments, the buffer comprises about 50 mMTrisHCl and about 500 mM NaCl with a pH of 8.5. In certain embodiments,the buffer comprises about 50 mM TrisHCl and about 750 mM NaCl with a pHof 8.0. In certain embodiments, the buffered solution comprises 45-55%(w/w) ethylene glycol.

In certain embodiments, each sugar solution comprises a disaccharide ortrisaccharide. In certain embodiments, the disaccharide comprisessucrose, maltose, lactose, and combinations thereof. In certainembodiments, each sugar solution comprises sucrose. In certainembodiments, each of the sugar solutions further comprises TrisHCl andNaCl. In certain embodiments, the TrisHCl is at a concentration of about20 to about 50 mM and the NaCl is at a concentration of about 150 mM toabout 500 mM. In certain embodiments, the buffered solution has a pH ofabout 7.4 to about 8.5. In certain embodiments, the buffer comprises 20mM TrisHCl and 8 g/L NaCl with a pH of 7.4.

In certain aspects, the methods comprise operating the ultracentrifugeat a first rotational speed of less than 10,000 rpm for less than 60minutes, and at a second rotational speed within the range of about30,000 to about 40,000 rpm for at least 4 hours.

In certain aspects, the methods comprise operating the ultracentrifugeat a first rotational speed of less than 10,000 rpm for less than 60minutes, and at a second rotational speed within the range of about30,000 to about 40,000 rpm for at least 12 hours.

In certain embodiments, the first rotational speed is about 3,000 rpm toabout 6,000 rpm, optionally, about 4,000 rpm. In certain embodiments,the second rotational speed is about 35,000 rpm and optionally ismaintained for about 4 to about 6 hours. In certain embodiments, thesecond rotational speed is maintained for at least about 16 hours or atleast 20 hours. In certain embodiments, the second rotational speed isabout 35,000 rpm and optionally is maintained for about 16 to about 20hours.

In certain embodiments, the concentrated AAV fraction loaded into thezonal rotor comprises at least 1×10¹² AAV capsids per mL. In certainembodiments, the methods comprise harvesting a supernatant from a cellculture comprising HEK293 cells transfected with a triple plasmidsystem. In certain embodiments, the methods comprise (i) harvesting thesupernatant about 3 to about 5 days after transfection of the HEK293cells or (ii) when the cell culture has a cell density of greater thanor about 5×10⁶ cells/mL and has a cell viability of greater than 50%. Incertain embodiments, the methods comprise filtering the harvestedsupernatant via depth filtration. In certain embodiments, the methodscomprise filtering the harvested supernatant through a filter comprisingcellulose and perlites and having a minimum permeability of about 500L/m². In certain embodiments, the methods comprise filtering theharvested supernatant through a filter with a minimum pore size of about0.2 μm. In certain embodiments, the methods comprise concentrating anAAV fraction using an ultra/diafiltration system. In certainembodiments, the methods comprise concentrating an AAV fraction using anultra/diafiltration system before, after, or before and after a stepcomprising applying an AAV fraction to an anion exchange (AEX)chromatography column under conditions that allow for the AAV to flowthrough the AEX chromatography column. In certain embodiments, themethods comprise inactivating lipid enveloped viruses of an AAV fractionwith a solvent detergent. In certain embodiments, the methods comprisenanofiltration of an AAV fraction to remove viruses greater than 35 nm.In certain embodiments, the methods comprise a polish step comprisingperforming AEX chromatography with a column comprising tentacle gel.

In certain embodiments, the methods comprise (i) applying an AAVfraction to an anion exchange (AEX) chromatography column underconditions that allow for the AAV to flow through the AEX chromatographycolumn and (ii) collecting the flow-through comprising the AAV. Incertain embodiments, the AAV fraction is applied to the AEXchromatography column with a loading buffer comprising about 100 mM toabout 150 mM NaCl, optionally, wherein the pH of the loading buffer isabout 8 to about 9. In certain embodiments, the loading buffer comprisesabout 115 mM to about 130 mM NaCl, optionally, the loading buffercomprises about 120 mM to about 125 mM NaCl.

In certain embodiments, host cell proteins are removed. In certainembodiments, host cell proteins are HSP70 and/or LDH.

In exemplary aspects, at least or about 55% of the AAV particles in thefraction obtained from the sugar gradient are full AAV capsids. Inexemplary aspects, greater than or about 60% of the AAV particles in thefraction obtained from the sugar gradient are full AAV capsids. Inexemplary aspects, greater than or about 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% of the AAV particles in the fractionobtained from the sugar gradient are full AAV capsids.

In certain embodiments, the methods comprise testing an AAV fraction viaan AAV-specific ELISA. In certain embodiments, the AAV specific ELISA isa sandwich ELISA specific for AAV. In certain embodiments, the methodsdo not include a step of measuring potency via quantitative PCR.

In certain embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, or AAV10. In certain embodiments, the AAV is AAV8.

Provided herein are also AAV products produced by the methods describedabove and herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of an exemplary method of the present disclosure.

FIG. 2 is a graph of the amount of virus particles (viral grams) per mlof supernatant of a transfected cell culture plotted over a time course(days). Propagation of AAV8 in HEK293 cell cultures under establishedstandard conditions (e.g. pH, Temp, agitation rate) in 2 L scale (4parallel runs with two different PEI lots): increase of titer in cellculture supernatant over time measured by FIX specific qPCR(T01=transfection day 1, T02=transfection 2, etc.). The titer wasmeasured by FIX specific qPCR in 3×2 L scale bioreactors.

FIG. 3 is a graph of the amount of virus particles (viral grams) per mlof supernatant of a transfected cell culture plotted over a time course(days). Propagation of AAV8 in HEK293 cell cultures under establishedstandard conditions (e.g. pH, Temp, agitation rate) in 2 L scale (4parallel runs with two different PEI lots): increase of titer in cellculture supernatant over time measured by an AAV-specific ELISA(T01=transfection day 1, T02=transfection 2, etc.). The vector genometiter was measured by an AAV-specific ELISA in 3×2 L scale bioreactors.

FIG. 4 is a graph of the pressure and flow rate during the depthfiltration step, as measured continuously via two pressure sensors(pressure 1 before depth filtration, pressure 2 before membrane filterafter depth filter; flow=flow rate as measured with flow meter).

FIG. 5 is a graph of the flow through product of an AEX Mustang Qchromatography in negative (non-binding) mode.

FIG. 6 is a silver stain (left) and a western blot (right) of thefractions indicated.

FIG. 7 is a silver stain (left) and a western blot (right) of thefractions indicated.

FIG. 8 is a graph of the ultracentrifugation elution profile separatingthe empty versus full capsids. The fractions were each 50 ml.

FIG. 9 is a silver stain (left) and a western blot (right) of thefractions indicated.

FIG. 10 is the final polishing of AAV8 on Fractogel TMAE.

FIG. 11 is a schematic of an exemplary method of the present disclosure.

FIG. 12 is a curve showing ultracentrifugation of separated full andempty AAV8 particles using the 50-55-60% protocol in aqueous solution,including particles containing different DNA sequence variants. TheVectorDNA was Human Coagulation Factor IX Padua, double stranded, Fulllength approx. 4.8 kB. Single fractions as indicated in the UC profilewere processed on a 4 ml AEX-TMAE column and formulated to a finalproduct. Each of the formulated fraction was analyzed. Fraction 10 to 14were pooled and represents the homologous end product that contains AAV8with full length vector DNA. Fraction 15 to 21 and additionally fraction25 and 26 are processed separately and show decreasing length of thevector DNA encapsulated in the AAV8 particles. This is shown in thexDNA-Agarose gels of FIGS. 14 and 15 and in the data of the analyticalultracentrifugation (AUC) shown in FIG. 16.

FIG. 13 is a silver stain (left) and western blot (right) of thefractions indicated.

FIG. 14 is a photograph of a 1% agarose gel of the following samplestaken from the ultracentrifugation of AAV8 particles described inExample 9: Lane 1=Marker; Lane 2=PV007 (AAV8 derived from the originalChatham process); Lane 3=Fraction 15; Lane 4=Fraction 16; Lane5=Fraction 17; Lane 6=Fraction 18; Lane 7=Fraction 19; Lane 8=Fraction20; Lane 9=Fraction 21; Lane 10=Fraction 25; Lane 11=Fraction 26; Lane12=product AAV8 for clinical administration; and Lane 13=Marker.BDS=bulk drug substance (final diluted TMAE-Eluate). This figure showsthat pooled fractions 10 to 14 of the UV peak obtained at higher sucrosedensity in the ultracentrifugation contain a highly pure single DNA bandwithout any further hint to smaller DNA variants of the vector DNA.

FIG. 15 is a photograph of a 0.8% alkaline agarose gel of the followingsamples taken from the ultracentrifugation of AAV8 particles describedin Example 9: Lane 1=Marker; Lane 2=PV007 (AAV8 derived from previousproduction processes); Lane 3=Fraction 15; Lane 4=Fraction 16; Lane5=Fraction 17; Lane 6=Fraction 18; Lane 7=Fraction 19; Lane 8=Fraction20; Lane 9=Fraction 21; Lane 10=Fraction 25; Lane 11=Fraction 26; Lane12=product AAV8 for clinical administration; and Lane 13=Marker.BDS=bulk drug substance (final diluted TMAE-Eluate). This figure showsthat pooled fractions 10 to 14 of the UV peak obtained at higher sucrosedensity in the ultracentrifugation contain a highly pure single DNA bandwithout any further hint to smaller DNA variants of the vector DNA.

FIG. 16A-16B represents the Fractogel TMAE elution profiles derived fromthe indicated fractions of the analytical ultracentrifugation (AUC) asstarting material. The full and empty capsids are identified witharrows. A subpopulation of capsids containing incomplete vector DNA(also defined herein as empty) is also identified. Data are alsorepresented in Table 17. See FIG. 12 regarding the fractions.

FIG. 17 is a graph of the ultracentrifugation elution profile in whichAAV8 in a 50% (w/w) ethylene glycol buffered solution in Tris/NaCl isultracentrifuged for 20 hours at 35,000 rpm in using a sucrose gradientwith 55% and 60% sucrose solutions. The ratio of AAV8 loading sample tothe sucrose gradient is 1:1. The vector DNA is human coagulation FactorIX Padua, single stranded self-complementary, full length (2.6 kB).

FIG. 18 is a graph of a separation of AAV8 containing single strandedvector DNA of human coagulation Factor IX Padua (2.6 kB) usingultracentrifugation with the 55-60% sugar layer protocol, where in theAAV8 sample is loaded in a 50% (w/w) ethylene glycol buffered solutionin Tris/NaCl. Each Fraction is tested via: AAV8 ITR qPCR, AAV8 Antigen,WAX (Weak anion exchanger—full capsids), DNA/AAV ratio is a ratio of ITRqPCR vector genomes (vg/ml)/AAV8 capsid antigen ELISA (cp/ml). The datawas normalized to allow the graphs to be presented on the same axes.

FIG. 19 is a graph of the ultracentrifugation elution profile in whichAAV8 in a 50% (w/w) ethylene glycol in Tris/NaCl buffer isultracentrifuged for 20 hours at 35000 rpm in through a sucrose gradientwith 55% and 60% sucrose solutions. The ratio of AAV8 loading sample tothe sucrose gradient is 1:1, with a core volume of 3,200 ml. The vectorDNA is human coagulation Factor VIII, full length (˜4.8 kB).

FIG. 20 is an overlay of sedimentation coefficient graphs from Fractions8-10 (see FIG. 19) demonstrating subspecies separation. The arrowdenotes a shift towards AAV8 with lower weight from fractions withhigher density to lower density in the UC-gradient. Fraction 8>Fraction9>Fraction 10.

FIG. 21 is a graph of a separation of AAV8 containing single strandedvector DNA of human coagulation Factor IX Padua (2.6 kB) usingultracentrifugation with the 50-55-60% sugar layer protocol, where theAAV8 sample is loaded in a TrisHCl/NaCl buffered solution. Each Fractionis tested via: AAV8 ITR qPCR and ELISA. DNA/AAV ratio is a ratio of ITRqPCR vector genomes (vg/ml)/AAV8 capsid antigen ELISA (cp/ml). The datawas normalized to allow the graphs to be presented on the same axes.

FIG. 22 is a graph of the ultracentrifugation elution profile in whichAAV8 in a 50% (w/w) ethylene glycol in Tris/NaCl buffer isultracentrifuged for 20 hours at 35000 rpm in through a sucrose gradientwith 55% and 60% sucrose solutions. The ratio of AAV8 loading sample tothe sucrose gradient is 1:1, with a core volume of 7,700 ml. The vectorDNA is human coagulation Factor IX Padua, single strandedself-complementary, full length (˜2.6 kB).

DETAILED DESCRIPTION

Provided herein are methods of producing an adeno-associated virus (AAV)product, methods of purifying AAV, and methods of purifying full AAVcapsids from a concentrated AAV fraction comprising empty AAV capsidsand full AAV capsids.

Advantageously, the methods are scalable to large volumes of startingmaterial, e.g., cell culture. In certain embodiments, the methodsprovided herein are large-scale methods capable of purifying AAV fromvolumes of at least or about 150 L, at least or about 250 L, at least orabout 500 L, at least or about 600 L, at least or about 700 L, at leastor about 800 L, at least or about 900 L, or at least or about 1000 L. Incertain embodiments, the methods are scalable to a minimum volume ofstarting material (e.g., cell culture) of at least or about 1250 L, atleast or about 1500 L, at least or about 2000 L, at least or about 2500L, at least or about 3000 L, at least or about 4000 L, at least or about5000 L, at least or about 6000 L, at least or about 7000 L, at least orabout 8000 L, at least or about 9000 L, at least or about 10,000 L, ormore. For example, the methods are carried out with a minimum volume ofabout 1000 L or about 10,000 L or 25,000 L or more cell cultureproducing AAV.

The methods of producing and purifying AAV described herein are alsoadvantageous, because the methods result in high titer AAV production.In certain embodiments, an AAV product comprising at least about 10¹⁰virus particles (vp) is produced from about 1000 L of starting material(e.g., cell culture). In certain embodiments, an AAV product comprisingat least about 10¹¹ virus particles (vp) is produced from about 1000 Lof starting material (e.g., cell culture). In certain embodiments, anAAV product comprising at least about 10¹² virus particles (vp) isproduced from about 1000 L of starting material (e.g., cell culture). Incertain embodiments, an AAV product comprising at least about 10¹³ virusparticles (vp) is produced from about 1000 L of starting material (e.g.,cell culture). In certain embodiments, an AAV product comprising atleast about 10¹⁴ virus particles (vp) is produced from about 1000 L ofstarting material (e.g., cell culture). In certain embodiments, an AAVproduct comprising at least about 10¹⁵ virus particles (vp) is producedfrom about 1000 L of starting material (e.g., cell culture). In certainembodiments, an AAV product comprising at least about 2×10¹⁵ virusparticles (vp) is produced from about 1000 L of starting material (e.g.,cell culture). In certain embodiments, an AAV product comprising atleast about 5×10¹⁵ virus particles (vp) is produced from about 1000 L ofstarting material (e.g., cell culture).

The methods of the present disclosure which provide high yields of AAV,include, in certain embodiments, a nanofiltration step to remove viruseslarger than the exclusion specification of the filter used in thenanofiltration step. This filtration step allows for the final AAVproduct to be a safer product for human administration, compared tothose AAV products achieved by other methods. The nanofiltration step isan effective virus reduction step for viruses larger than the exclusionspecification of the filter used in the nanofiltration step, which, incertain embodiments, means that the method has the ability to removemore than 4 logs of contaminant viruses from the AAV product viananofiltration, as further described herein.

Another advantage of the methods described herein is that the methodsyield a highly pure AAV product. In certain embodiments, the AAV productproduced through the methods of the present disclosure is substantiallyfree of one or more contaminants: host cell proteins, host cell nucleicacids (e.g., host cell DNA), plasmid DNA, empty viral vectors (includingcontaining truncated or incomplete vector DNA), AAV particles withincomplete protein composition and oligomerized structures, orcontaminating viruses, e.g., non AAV, lipid enveloped viruses, Heatshock protein 70 (HSP70), Lactate dehydrogenase (LDH), proteasomes,contaminant non-AAV viruses (e.g., lipid-enveloped viruses), host cellculture components (e.g., peptides, antibiotics), process relatedcomponents (e.g. 0.3% Tri-n-butylphosphate; 1.0% Triton X100,polyethylenimine), mycoplasma, pyrogens, bacterial endotoxins, andadventitious agents. AAV products can be determined to be substantiallyfree if they appear free of the impurity as determined by standardmethods of analysis, such as, but not limited to, thin layerchromatography (TLC), gel electrophoresis and high performance liquidchromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), or ITRqPCR, used by those of skill in the art to assess such purity, orsufficiently pure such that further purification would not detectablyalter the physical and chemical properties, such as enzymatic andbiological activities, of the substance. In one embodiment, the term“substantially free of an impurity” includes preparations of AAV havingless than about 50% (by dry weight), 45%, 40%, 35%, 30%, 25%, 20%, 15%,10%, 5%, 4%, 3%, 2%, or 1% of non-full capsid AAV material. In anotherembodiment, the term “substantially free of an impurity” includespreparations of AAV wherein the impurity represents less than about orat 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% ofthe volume of the AAV product.

In exemplary embodiments, the methods of the present disclosure providea purified AAV product wherein at least or about 50% of the contaminantfound in the starting material (e.g., cell culture) is removed. Inexemplary embodiments, the methods of the present disclosure provide apurified AAV product wherein at least or about 60% of the contaminantfound in the starting material (e.g., cell culture) is removed. Inexemplary embodiments, the methods of the present disclosure provide apurified AAV product wherein at least or about 70% of the contaminantfound in the starting material (e.g., cell culture) is removed. Inexemplary embodiments, the methods of the present disclosure provide apurified AAV product wherein at least or about 80% of the contaminantfound in the starting material (e.g., cell culture) is removed. Inexemplary embodiments, the methods of the present disclosure provide apurified AAV product wherein at least or about 90% of the contaminantfound in the starting material (e.g., cell culture) is removed.

In certain embodiments, the AAV product produced through the methods ofthe present disclosure is suitable for administration to a human. Incertain embodiments, the AAV is a recombinant AAV (rAAV). In certainembodiments, the AAV product produced through the methods of the presentdisclosure is sterile and/or of good manufacturing practice (GMP) grade.In certain embodiments, the AAV product produced through the methods ofthe present disclosure conforms to the requirements set forth in theU.S. Pharmacopeia Chapter 1046 or the European Pharmacopoeia on genetherapy medicinal products or as mandated by the U.S. Food and DrugAdministration (USFDA) or the European Medicines Agency (EMA).

Additionally, the AAV products produced from the methods describedherein are highly potent. The potency of an AAV product, e.g., AAV8product, can be described in terms of (1) in vivo biopotency (e.g.,production of active protein in mice) which is given as units (FIX orFVIII) per mL of mouse plasma; or (2) in vitro biopotency. The in vitrobiopotency test measures the potential of AAV vectors to transducecells, e.g., HepG2 cells, which express and secrete the protein ofinterest into the medium, and determine the amount by ELISA techniquesand/or enzyme activity. Suitable methods of measuring in vivo and invitro biopotency are known in the art and are also described herein asExample 12.

In further embodiments, the AAV product produced from the methodsdescribed herein demonstrate superior DNA/AAV ratio. In exemplaryembodiments, the AAV product produced from the methods described hereindemonstrate a superior ratio of vector genomes per μg of AAVdemonstrating that the AAV product has a high amount of full virusparticles. In certain embodiments, the methods of the present disclosurecomprise testing an AAV fraction via an AAV-specific ELISA. In certainembodiments, the AAV-specific ELISA is sufficient to provide arepresentative reading on potency of the AAV fraction, because themajority of the capsids in the AAV fraction are full capsids.

Sugar Gradient Ultracentrifugation

In exemplary embodiments, the methods of the present disclosure comprisean ultracentrifugation step during which a density gradient is formed.Though not wishing to be bound to a theory, it is believed that theultracentrifugation step allows for full AAV capsids to be separatedfrom empty AAV capsids. As used herein, the term “full AAV capsids” withregard to AAV or AAV capsids or AAV particles refer to those containingthe complete vector genome. Full AAV capsids can provide a therapeuticbenefit to recipient patients. As used herein, the term “empty” withregard to AAV or AAV capsids or AAV particles refer to those that lackthe complete (i.e., full) vector genome. In certain embodiments, “empty”can also include “incomplete vector DNA” or “truncated vector DNA”. Suchempty AAV or empty AAV capsids or empty AAV particles may lack thevector genome in part or in whole, i.e., they may be partially empty orcompletely empty, and, as such, are unable to provide a therapeuticbenefit.

Accordingly, the present disclosure provides a method of separating fullAAV capsids from empty AAV capsids or a method of purifying full AAVcapsids from a concentrated AAV fraction comprising full AAV capsids andempty AAV capsids. In exemplary aspects, the methods of the presentdisclosure comprise (i) loading into a rotor an AAV fraction (e.g., asolution comprising AAV) with at least two sugar solutions, each sugarsolution of which has a different sugar concentration, (ii) operating anultracentrifuge comprising the loaded rotor in batch mode to form asugar gradient, and (iii) obtaining a fraction of the sugar gradient toobtain an AAV fraction comprising full AAV capsids. In certainembodiments, at least three, at least four, at least five, or at leastsix sugar solutions are loaded. In certain embodiments, two sugarsolutions are loaded. In certain embodiments, three sugar solutions areloaded.

The order of loading sequence can be first the AAV fraction (e.g., asolution comprising AAV) followed by the lowest concentration of sugarsolution, then followed by the next more concentrated sugar solution,and so on.

In exemplary aspects, the method comprises loading into a rotor an AAVfraction (e.g., a solution comprising AAV) with at least two sugarsolutions, each sugar solution of which (a) has a different sugarconcentration and (b) comprises a sugar at a concentration equivalent toa sucrose concentration ranging from about 45% (w/w) to about 65% (w/w)sucrose. In certain embodiments, the method comprises a sugar at aconcentration equivalent to a sucrose concentration ranging from about50% (w/w) to about 60% (w/w) sucrose or about 55% (w/w) to about 60%(w/w) sucrose.

In certain embodiments, at least one sugar solution comprises sugar at aconcentration equivalent to a sucrose concentration between 52-58% (w/w)sucrose, at least another sugar solution comprises sugar at aconcentration equivalent to a sucrose concentration between 57-63% (w/w)sucrose, and optionally, another sugar solution comprising a sugar at aconcentration equivalent to a sucrose concentration between 47-53% (w/w)sucrose.

In certain embodiments, at least one sugar solution comprises sugar at aconcentration equivalent to a sucrose concentration between 54-56% (w/w)sucrose, at least another sugar solution comprises sugar at aconcentration equivalent to a sucrose concentration between 59-61% (w/w)sucrose, and optionally, another sugar solution comprising a sugar at aconcentration equivalent to a sucrose concentration between 49-51% (w/w)sucrose.

In certain embodiments, at least one sugar solution comprises sugar at aconcentration equivalent to a sucrose concentration greater than about54% (w/w) sucrose, at least another sugar solution comprises sugar at aconcentration equivalent to a sucrose concentration greater than about59% (w/w) sucrose, and optionally, another sugar solution at aconcentration equivalent to a sucrose concentration greater than about49% (w/w) sucrose.

In certain embodiments, at least one sugar solution comprises sugar at aconcentration equivalent to a sucrose concentration equal to or greaterthan about 55% (w/w) sucrose, at least another sugar solution comprisessugar at a concentration equivalent to a sucrose concentration equal toor greater than about 60% (w/w) sucrose, and optionally, another sugarsolution at a concentration equivalent to a sucrose concentration equalto or greater than about 50% (w/w) sucrose.

In certain embodiments, at least one sugar solution comprises sugar at aconcentration equivalent to a sucrose concentration of about 55% (w/w)sucrose, and at least another sugar solution comprises sugar at aconcentration equivalent to a sucrose concentration of about 60% (w/w)sucrose, and optionally, another sugar solution at a concentrationequivalent to a sucrose concentration of about 50% (w/w) sucrose.

The order of loading sequence can be first the AAV fraction (e.g., asolution comprising AAV) followed by the lowest concentration of sugarsolution, then followed by the next more concentrated sugar solution(e.g., first the sugar solution comprising a sugar at a concentrationequivalent to a sucrose concentration between 47-53% (w/w) sucrose, ifpresent, then the sugar solution comprising a sugar at a concentrationequivalent to a sucrose concentration between 52-58% (w/w) sucrose, andthen the sugar solution comprising a sugar at a concentration equivalentto a sucrose concentration between 57-63% (w/w) sucrose).

An advantage of embodiments with at least one sugar solution comprisingsugar at a concentration equivalent to a sucrose concentration of 52-58%(w/w) sucrose, 53-57% (w/w) sucrose or about 55% (w/w) sucrose, and atleast another sugar solution comprises sugar at a concentrationequivalent to a sucrose concentration of 57-63% (w/w) sucrose, 58-62%(w/w) sucrose or about 60% (w/w) sucrose, is that gradients formed ofsuch sugar solutions can provide improved separation of AAV capsids.

In exemplary aspects, the method comprises loading into a rotor an AAVfraction (e.g., a solution comprising AAV) with at least three sugarsolutions, each sugar solution of which (a) has a different sugarconcentration and (b) comprises a sugar at a concentration equivalent toa sucrose concentration ranging from about 45% (w/w) to about 65% (w/w)sucrose. In certain embodiments, the method comprises a sugar at aconcentration equivalent to a sucrose concentration ranging from about50% (w/w) to about 60% (w/w) sucrose.

In certain embodiments, at least one sugar solution comprises sugar at aconcentration equivalent to a sucrose concentration between 47-53% (w/w)sucrose, at least another sugar solution comprises sugar at aconcentration equivalent to a sucrose concentration between 52-58% (w/w)sucrose, and at least another sugar solution comprises sugar at aconcentration equivalent to a sucrose concentration between 57-63% (w/w)sucrose.

In certain embodiments, at least one sugar solution comprises sugar at aconcentration equivalent to a sucrose concentration between 49-51% (w/w)sucrose, at least another sugar solution comprises sugar at aconcentration equivalent to a sucrose concentration between 54-56% (w/w)sucrose, and at least another sugar solution comprises sugar at aconcentration equivalent to a sucrose concentration between 59-61% (w/w)sucrose.

In certain embodiments, at least one sugar solution comprises sugar at aconcentration equivalent to a sucrose concentration greater than about49% (w/w) sucrose, at least another sugar solution comprises sugar at aconcentration equivalent to a sucrose concentration greater than about54% (w/w) sucrose, and at least one sugar solution at a concentrationequivalent to a sucrose concentration greater than about 49% (w/w)sucrose.

In certain embodiments, at least one sugar solution comprises sugar at aconcentration equivalent to a sucrose concentration equal to or greaterthan about 50% (w/w) sucrose, at least another sugar solution comprisessugar at a concentration equivalent to a sucrose concentration equal toor greater than about 55% (w/w) sucrose, and at least one sugar solutionat a concentration equivalent to a sucrose concentration equal to orgreater than about 60% (w/w) sucrose.

In certain embodiments, at least one sugar solution comprises sugar at aconcentration equivalent to a sucrose concentration of about 50% (w/w)sucrose, at least one sugar solution comprises sugar at a concentrationequivalent to a sucrose concentration of about 55% (w/w) sucrose, and atleast another sugar solution comprises sugar at a concentrationequivalent to a sucrose concentration or about 60% (w/w).

In certain embodiments, the order of loading sequence can be first, thesugar solution comprising comprises a sugar at a concentrationequivalent to a sucrose concentration between 47-53% (w/w) sucrose, thenthe sugar solution comprising comprises a sugar at a concentrationequivalent to a sucrose concentration between 52-58% (w/w) sucrose, andthen the sugar solution comprises a sugar at a concentration equivalentto a sucrose concentration between 57-63% (w/w) sucrose.

The order of loading sequence can be first the AAV fraction (e.g., asolution comprising AAV) followed by the lowest concentration of sugarsolution, then followed by the next more concentrated sugar solution(e.g., first the sugar solution comprising a sugar at a concentrationequivalent to a sucrose concentration between 47-53% (w/w) sucrose, thenthe sugar solution comprising a sugar at a concentration equivalent to asucrose concentration between 52-58% (w/w) sucrose, and then the sugarsolution comprising a sugar at a concentration equivalent to a sucroseconcentration between 57-63% (w/w) sucrose).

In exemplary aspects, each of the AAV fraction and/or sugar solutionsmay comprise additional components, e.g., buffering agents, salts, andthe like. In certain embodiments, each of the AAV fraction and/or sugarsolutions comprises, individually, any buffer substance or combinationsthereof to stabilize the pH from about 5.5 to about 8.5. Examples ofacceptable buffering agents are well known in the art, and include,without limitation, phosphate buffers, histidine (e.g., L-histidine),sodium citrate, HEPES, Tris, Bicine, glycine, N-glycylglycine, sodiumacetate, sodium carbonate, glycyl glycine, lysine, arginine, sodiumphosphate, and mixtures thereof. In certain embodiments, the buffer isTrisHCl or TrisHCl/NaCl. The pH of the buffer/fraction/solution may be5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0. The pH of thebuffer/fraction/solution may be from 7.0 to 9.0, from 7.1 to 8.9, from7.2 to 9.0, from 7.0 to 8.8, from 7.2 to 8.8, from 7.1 to 8.6, from 7.3to 8.9, from 7.4 to 9.0, or from 7.4 to 8.5. In certain embodiments,each, individually, of the buffer, AAV fraction and/or sugar solutionshave a pH of about 7.4. In certain embodiments, each, individually, ofthe buffer, AAV fraction and/or sugar solutions have a pH of about 8.0.In certain embodiments, each, individually, of the buffer, AAV fractionand/or sugar solutions have a pH of about 8.5.

In certain embodiments, the buffer can be TrisHCl/NaCl. In certainembodiments, TrisHCl is at a concentration of about 10 mM to about 100mM or about 20 to about 50 mM. In certain embodiments, the TrisHCl is ata concentration of about 10 mM, about 20 mM, about 25 mM, about 30 mM,about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 75 mM, about80 mM, about 90 mM, or about 100 mM. In certain embodiments, NaCl is ata concentration of about 100 mM to about 1 M, about 150 mM to about 750mM, about 150 mM to about 500 mM, or about 500 mM to about 750 mM. Incertain embodiments, the concentration of NaCl is about 100 mM, about120 mM, about 125 mM about 130 mM, about 136 mM, about 140 mM, about 150mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275mM, about 300 mM, about 325 mM, about 350 mM, about 375 mM, about 400mM, about 425 mM, about 450 mM, about 475 mM, about 500 mM, about 525mM, about 550 mM, about 575 mM, about 600 mM, about 625 mM, about 650mM, about 675 mM, about 700 mM, about 725 mM, about 750 mM, about 775mM, about 800 mM, about 825 mM, about 850 mM, about 875 mM, about 900mM, about 925 mM, about 950 mM, about 975 mM, or about 1 M. In certainembodiments, TrisHCl is at a concentration of about 10 mM to about 100mM or about 20 mM to about 50 mM and NaCl is at a concentration of about100 mM to about 1M, about 150 mM to about 750 mM, about 150 mM to about500 mM, or about 500 mM to about 750 mM. In certain embodiments, TrisHClis at a concentration of about 50 mM and NaCl is at a concentration ofabout 500 mM. In certain embodiments, TrisHCl is at a concentration ofabout 20 mM and NaCl is at a concentration of about 136 mM. In certainembodiments, TrisHCl is at a concentration of about 50 mM and NaCl is ata concentration of about 750 mM. In certain embodiments, the buffer is aTrisHCl/NaCl buffer with a pH or about 7.4. In certain embodiments, thebuffer is a TrisHCl/NaCl buffer with a pH or about 8.0. In certainembodiments, the buffer is a TrisHCl/NaCl buffer with a pH or about 8.5.

In certain embodiments, the AAV fraction solution comprises about 50 mMTrisHCl and 500 mM NaCl at a pH of 8.5. In certain embodiments, the AAVfraction solution comprises about 50 mM TrisHCl and 750 mM NaCl at a pHof 8.0. In certain embodiments, the sugar solutions comprise about 20 mMTrisHCl and 136 mM NaCl at a pH of 7.4.

In exemplary aspects, the method comprises loading into a rotor an AAVfraction (e.g., a solution comprising AAV) comprising a bufferedethylene glycol solution and at least two sugar solutions, each sugarsolution of which (a) has a different sugar concentration and (b)comprises a sugar at a concentration equivalent to a sucroseconcentration ranging from about 45% (w/w) to about 65% (w/w) sucrose.In certain embodiments, the method comprises a sugar at a concentrationequivalent to a sucrose concentration ranging from about 50% (w/w) toabout 60% (w/w) sucrose or about 55% (w/w) to about 60% (w/w) sucrose.The range of sugar concentrations are the same as listed above. Anadvantage of using the buffered ethylene glycol solution is that shallowgradients can be performed. Without wishing to be bound by theory, theethylene glycol solution may change the viscosity and/or densityproperties of the ultracentrifugation matrix. It was surprisingly foundthat the addition of ethylene glycol in combination with an increasedprocessing time creates a shallow gradient, which allows the separationof the full capsids from empty capsids containing smaller vector DNA(e.g., 3 kb or 5 kb) from the empty capsids. As used herein, a “shallowgradient” is one in which the solution density or thegradient-forming-solute concentration changes (e.g., sucrose gradient)gradually versus distance. This is opposed to one in which the solutiondensity or the gradient-forming-solute concentration changes rapidlyversus distance. For example, AAV with a DNA vector having about 2.5 toabout 5.0 kb can be resolved more efficiently when the loading buffercomprises about 45% to about 55% ethylene glycol solution. In certainembodiments, use of ethylene glycol in the AAV fraction solution allowsfor the separation of a DNA vector having between about 2.5 to about 3.0kb. In certain embodiments, the method allows for a greater resolutionof a DNA vector having about 2.5, about 2.6, about 2.7, about 2.8, about2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5,about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8,about 4.9, or about 5.0 kb. In certain embodiments, the enhancedresolution occurs with a sugar solution with at least three differentsugar concentrations. In certain embodiments, the enhanced resolutionoccurs with a sugar solution with at least two different sugarconcentrations. In certain embodiments, the enhanced resolution occurswith a sugar solution with two different sugar concentrations. Incertain embodiments, the enhanced resolution occurs when using one sugarsolution at a concentration equivalent to a sucrose concentration of52-58% (w/w) sucrose, 53-57% (w/w) sucrose or about 55% (w/w) sucrose,and at least another sugar solution comprises sugar at a concentrationequivalent to a sucrose concentration of 57-63% (w/w) sucrose, 58-62%(w/w) sucrose or about 60% (w/w) sucrose.

In exemplary aspects, the method comprises loading into a rotor an AAVfraction (e.g., a solution comprising AAV) with a buffered ethyleneglycol solution and at least three sugar solutions, each sugar solutionof which (a) has a different sugar concentration and (b) comprises asugar at a concentration equivalent to a sucrose concentration rangingfrom about 45% (w/w) to about 65% (w/w) sucrose. In certain embodiments,the method comprises a sugar at a concentration equivalent to a sucroseconcentration ranging from about 50% (w/w) to about 60% (w/w) sucrose.The range of sugar concentrations are the same as listed above.

The buffered ethylene glycol solution may comprise any buffer substanceor combinations thereof to stabilize the pH from about 5.5 to about 8.5.Examples of acceptable buffering agents are well known in the art, andinclude without limitation, phosphate buffers, histidine, sodiumcitrate, HEPES, Tris, Bicine, glycine, N-glycylglycine, sodium acetate,sodium carbonate, glycyl glycine, lysine, arginine, sodium phosphate,and mixtures thereof. In certain embodiments, the buffer is TrisHCl orTrisHCl/NaCl. The pH of the buffer/solution may be 5.5, 5.6, 5.7, 5.8,5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,8.7, 8.8, 8.9, or 9.0. The pH of the buffer/solution may be from 7.0 to9.0, from 7.1 to 8.9, from 7.2 to 9.0, from 7.0 to 8.8, from 7.2 to 8.8,from 7.1 to 8.6, from 7.3 to 8.9, from 7.4 to 9.0, or from 7.4 to 8.5.In certain embodiments, the buffer/solution has a pH of about 7.4. Incertain embodiments, the buffer/solution has a pH of about 8.0. Incertain embodiments, the buffer/solution has a pH of about 8.5.

In certain embodiments, the buffered ethylene glycol solution comprisesTrisHCl/NaCl. In certain embodiments, TrisHCl is at a concentration ofabout 10 mM to about 100 mM or about 20 to about 50 mM. In certainembodiments, the TrisHCl is at a concentration of about 10 mM, about 20mM, about 25 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM,about 70 mM, about 75 mM, about 80 mM, about 90 mM, or about 100 mM. Incertain embodiments, NaCl is at a concentration of about 100 mM to about1 M, about 150 mM to about 750 mM, or about 150 mM to about 500 mM. Incertain embodiments, the concentration of NaCl is about 100 mM, about120 mM, about 125 mM about 130 mM, about 136 mM, about 140 mM, about 150mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275mM, about 300 mM, about 325 mM, about 350 mM, about 375 mM, about 400mM, about 425 mM, about 450 mM, about 475 mM, about 500 mM, about 525mM, about 550 mM, about 575 mM, about 600 mM, about 625 mM, about 650mM, about 675 mM, about 700 mM, about 725 mM, about 750 mM, about 775mM, about 800 mM, about 825 mM, about 850 mM, about 875 mM, about 900mM, about 925 mM, about 950 mM, about 975 mM, or about 1 M. In certainembodiments, TrisHCl is at a concentration of about 10 mM to about 100mM or about 20 mM to about 50 mM and NaCl is at a concentration of about100 mM to about 1M, about 150 mM to about 750 mM, about 150 mM to about500 mM, or about 500 mM to about 750 mM. In certain embodiments, TrisHClis at a concentration of about 50 mM and NaCl is at a concentration ofabout 500 mM. In certain embodiments, TrisHCl is at a concentration ofabout 20 mM and NaCl is at a concentration of about 136 mM. In certainembodiments, TrisHCl is at a concentration of about 50 mM and NaCl is ata concentration of about 750 mM. In certain embodiments, the buffer is aTrisHCl/NaCl buffer with a pH or about 7.4. In certain embodiments, thebuffer is a TrisHCl/NaCl buffer with a pH or about 8.0. In certainembodiments, the buffer is a TrisHCl/NaCl buffer with a pH or about 8.5.

The buffered ethylene glycol solution may comprise from 45% to 55%(w/w), from 46% to 54% (w/w), from 45% to 53% (w/w), from 47% to 55%(w/w), or from 48% to 52% (w/w) of the buffer (e.g., TrisHCl/NaCl). Thebuffered ethylene glycol solution may comprise 40% (w/w), 41% (w/w), 42%(w/w), 43% (w/w), 44% (w/w), 45% (w/w), 46% (w/w), 47% (w/w), 48% (w/w),49% (w/w), 50% (w/w), 51% (w/w), 52% (w/w), 53% (w/w), 54% (w/w), 55%(w/w), 56% (w/w), 57% (w/w), 58% (w/w), 59% (w/w), or 60% (w/w) of thebuffer (e.g., TrisHCl/NaCl). The buffered ethylene glycol solution maybe aqueous.

In certain embodiments, the buffered ethylene glycol solution comprises45-55% (w/w) ethylene glycol, 50 mM TrisHCl, and 750 mM NaCl at a pH of8.0. In certain embodiments, the buffered ethylene glycol solutioncomprises 50% (w/w) ethylene glycol, 50 mM TrisHCl, and 750 mM NaCl at apH of 8.0.

In certain embodiments, the volume of the ultracentrifuge rotor core isfrom about 800 ml to about 9000 ml. In certain embodiments, the volumeof the ultracentrifuge rotor core is from about 800 ml to about 7700 ml.In certain embodiments, the volume of the ultracentrifuge rotor core isabout 800 ml. In certain embodiments, the volume of the ultracentrifugerotor core is about 1600 ml. In certain embodiments, the volume of theultracentrifuge rotor core is about 3200 ml. In certain embodiments, thevolume of the ultracentrifuge rotor core is about 7700 ml.

In certain embodiments, the sugar solutions are loaded with a constantflow rate. In certain embodiments, the constant flow rate is from about0.25 L/hr to about 10 L/hr or about 1.5 L/hr to about 7 L/hr. In certainembodiments, the constant flow rate is about 0.25 L/hr, about 0.5 L/hr,about 0.75 L/hr, about 1 L/hr, about 1.25 L/hr, about 1.5 L/hr, about1.75 L/hr, about 2 L/hr, about 2.5 L/hr, about 3 L/hr, about 3.5 L/hr,about 4 L/hr, about 4.5 L/hr, about 5 L/hr, about 5.5 L/hr, about 6L/hr, about 6.5 L/hr, about 7 L/hr, about 7.5 L/hr, about 8 L/hr, about8.5 L/hr, about 9 L/hr, about 9.5 L/hr, or about 10 L/hr.

In certain embodiments, the method comprises loading into a rotor about45-55% (w/w) of an AAV fraction (with or without a buffered ethyleneglycol solution) and about 45-55% (w/w) of the at least two sugarsolutions. In exemplary embodiments, the method comprises loading into arotor about 50% (w/w) of an AAV fraction (with or without a bufferedethylene glycol solution) and about 50% (w/w) of the at least two sugarsolutions. In certain embodiments, the volume of the ultracentrifugerotor core is from about 800 ml to about 3200 ml and the rotor is loadedwith about 45-55% (w/w) of an AAV fraction (with or without a bufferedethylene glycol solution) and about 45-55% (w/w) of the at least twosugar solutions. In certain embodiments, the volume of theultracentrifuge rotor core is from about 800 ml to about 3200 ml and therotor is loaded with about 50% (w/w) of an AAV fraction (with or withouta buffered ethylene glycol solution) and about 50% (w/w) of the at leasttwo sugar solutions in total. For example, if the rotor core is 800 ml,the AAV fraction can be 400 ml and the total sugar solution portion is400 ml.

In certain embodiments, the method comprises loading into a rotor about65-85% (w/w) of an AAV fraction (with or without a buffered ethyleneglycol solution) and 15-35% (w/w) of the at least two sugar solutions.In certain embodiments, the method comprises loading into a rotor about70-80% (w/w) of an AAV fraction (with or without a buffered ethyleneglycol solution) and about 20-30% (w/w) of the at least two sugarsolutions. In exemplary embodiments, the method comprises loading into arotor about 75% (w/w) of an AAV fraction (with or without a bufferedethylene glycol solution) and about 25% (w/w) of the at least two sugarsolutions. In exemplary embodiments, the method comprises loading into arotor about 74% (w/w) of an AAV fraction (with or without a bufferedethylene glycol solution) and about 26% (w/w) of the at least two sugarsolutions. In certain embodiments, the volume of the ultracentrifugerotor core is from about 7700 ml to about 9000 ml and the rotor isloaded with about 70-80% (w/w) of an AAV fraction (with or without abuffered ethylene glycol solution) and about 20-30% (w/w) of the atleast two sugar solutions. In certain embodiments, the volume of theultracentrifuge rotor core is from about 7700 ml to about 9000 ml andthe rotor is loaded with about 75% (w/w) of an AAV fraction (with orwithout a buffered ethylene glycol solution) and about 25% (w/w) of theat least two sugar solutions. In certain embodiments, the volume of theultracentrifuge rotor core is from about 7700 ml to about 9000 ml andthe rotor is loaded with about 74% (w/w) of an AAV fraction (with orwithout a buffered ethylene glycol solution) and about 26% (w/w) of theat least two sugar solutions. For example, if the rotor core is 7700 ml,the AAV fraction can be 6100 ml and the total sugar solution portion canbe 1600 ml.

In certain embodiments, the sugar solutions are added at roughly thesame volumes. In certain embodiments, the method comprises loading atleast two sugar solutions, and the sugar solution with the smallestsugar concentration is loaded in the rotor at a volume which is equal tothe volume of the other sugar solutions in the rotor. In certainembodiments, the method comprises loading at least two sugar solutions,and the sugar solution with the smallest sugar concentration is loadedin the rotor at a volume which is twice the volume of one of the othersugar solutions in the rotor. In certain embodiments, the methodcomprises loading at least two sugar solutions, and the sugar solutionwith the smallest sugar concentration is loaded in the rotor at a volumewhich is about 1.6 times or about 2.6 times the volume of one of theother sugar solutions in the rotor. In certain embodiments, the methodcomprises loading at least two sugar solutions, and the sugar solutionwith the smallest sugar concentration is loaded in the rotor at a volumewhich is half the volume of one of the other sugar solutions in therotor. In certain embodiments, the sugar solution with the smallestsugar concentration is loaded in the rotor at a volume which is twicethe volume of the sugar solution with the largest sugar concentration,optionally, wherein the volume of the sugar solution with the largestsugar concentration is equal to the volume of the sugar solution withthe intermediate sugar concentration. In certain embodiments, the sugarsolution with the smallest sugar concentration is loaded in the rotor ata volume which is about 2.6 times the volume of the sugar solution withthe largest sugar concentration, optionally, wherein the volume of thesugar solution with the intermediate sugar concentration is about 1.6times the volume of the sugar solution with the largest sugarconcentration. In certain embodiments, the sugar solution with thesmallest sugar concentration is loaded in the rotor at a volume which isequal to the volume of the sugar solution with the largest sugarconcentration, optionally, wherein the volume of the sugar solution withthe largest sugar concentration is half the volume of the sugar solutionwith the intermediate sugar concentration. In certain embodiments, thesugar solution with the smallest sugar concentration is loaded in therotor at a volume which is half the volume of the AAV fraction. Incertain embodiments, the sugar solution with the smallest sugarconcentration is loaded in the rotor at a volume which is three-quartersthe volume of the AAV fraction. In certain embodiments, the ratio of thevolume of the sugar solutions to the volume of the AAV fraction is lessthan or equal to one.

In certain embodiments, the method comprises loading two sugar solutionsand the sugar solution with the smallest sugar concentration is loadedin the rotor at a volume which is equal to the volume of the largestsugar solution in the rotor. In certain embodiments, the sugar solutionwith the smallest sugar concentration is loaded in the rotor at a volumewhich is half the volume of the AAV fraction. In certain embodiments,the ratio of the volume of the sugar solutions to the volume of the AAVfraction is less than or equal to one.

In certain embodiments, the method comprises loading three sugarsolutions and the sugar solution with the smallest sugar concentrationis loaded in the rotor at a volume which is at least twice the volume ofone of the other two sugar solutions in the rotor. In certainembodiments, the sugar solution with the smallest sugar concentration isloaded in the rotor at a volume which is twice the volume of the sugarsolution with the largest sugar concentration, optionally, wherein thevolume of the sugar solution with the largest sugar concentration isequal to the volume of the sugar solution with the intermediate sugarconcentration. For example, the sugar solution with the smallest sugarsolution can be at a volume of about 750 ml to about 900 ml or about 800ml, the sugar solution with an intermediate sugar concentration can beat a volume of about 350 ml to about 450 ml or about 400 ml, and thesugar solution with the largest sugar concentration can be at a volumeof about 350 ml to about 450 ml or about 400 ml. In certain embodiments,the sugar solution with the smallest sugar concentration is loaded inthe rotor at a volume which is half the volume of the AAV fraction. Incertain embodiments, the sugar solution with the smallest sugarconcentration is loaded in the rotor at a volume which is three-quartersthe volume of the AAV fraction. In certain embodiments, the ratio of thevolume of the sugar solutions to the volume of the AAV fraction is lessthan or equal to one. In exemplary aspects, the rotor is a zonal rotor.In certain embodiments, the total volume of the sugar solutions and theAAV fraction is less than or equal to the volume of the zonal rotor. Incertain embodiments, the volume of the total volume of the solutions inthe zonal rotor is about 800 ml to 9 L. In certain embodiments, thevolume of the sugar solutions is greater than or equal to about 50% ofthe volume of the zonal rotor. For example, the volume of the sugarsolutions is greater than or equal to about 50% of the volume of a zonalrotor having a volume of less than about 3200 ml, e.g., about 3200 ml,about 1600 ml, or about 800 ml. In certain embodiments, the volume ofthe sugar solutions is less than or equal to about 25% of the volume ofthe zonal rotor, e.g., when a core of greater than 7 L, e.g., 7.7 L, isused.

In certain embodiments, the ratio of the volume of the total sugargradient to volume of the AAV fraction loaded in the zonal rotor is fromabout 1:1 to about 1:5. In some embodiments, the volume of the totalsugar gradient to volume of the AAV fraction loaded in the zonal rotoris about 1:1, about 1:1.25, about 1:1.5, about 1:1.75, about 1:2, about1:2.25, about 1:2.5, about 1:2.75, about 1:3, about 1:3.25, about 1:3.5,about 1:3.75, about 1:4, about 1:4.25, about 1:4.5, about 1:4.75, orabout 1:5.

In exemplary aspects, the method comprises loading into a zonal rotorthe concentrated AAV fraction with at least two sugar solutions, each ofwhich has a different sugar concentration and each of which comprises asugar at a concentration equivalent to a sucrose concentration rangingfrom about 45% (w/w) to about 65% (w/w) sucrose (ranges as outlinedabove), wherein (A) the volume of the sugar solutions is greater than orequal to about 50% of the volume of the zonal rotor, (B) the totalvolume of the sugar solutions and the AAV fraction is less than or equalto the volume of the zonal rotor, (C) the ratio of the volume of thesugar solutions to the volume of the AAV fraction is less than or equalto one, or (D) a combination of (A), (B) and (C). In certainembodiments, the volume of the total volume of the solutions in thezonal rotor is about 800 ml to 9 L. In certain embodiments, the volumeof the sugar solutions is greater than or equal to about 50% of thevolume of the zonal rotor. For example, the volume of the sugarsolutions is greater than or equal to about 50% of the volume of a zonalrotor having a volume of less than about 3200 ml, e.g., about 3200 ml,about 1600 ml, or about 800 ml. In certain embodiments, the volume ofthe sugar solutions is less than or equal to about 25% of the volume ofthe zonal rotor, e.g., when a core of greater than 7 L, e.g., 7.7 L, isused.

In exemplary aspects, each sugar solution comprises a solublecarbohydrate or a carbohydrate mixture. In certain embodiments, eachsugar solution comprises a disaccharide (e.g., sucrose, maltose orlactose) and/or a trisaccharide. In certain embodiments, the density ofthe sugar solutions are equal to the density of sucrose solutions with aconcentration ranging from about 45% (w/w) to about 65% (w/w). Incertain embodiments, at least one of the sugar solutions has a densitywhich is equal to about 60% sucrose at a given temperature. In certainembodiments, the ranges are as recited above.

For purposes herein, a sugar other than sucrose may be used providedthat the sugar in the sugar solution has a concentration equivalent to asucrose concentration within the specified range or at the specificamount (% (w/w)). The concentration of sucrose can be determined by arefractive index method, which determines the sugar content of anaqueous solution in degrees Brix (“° Bx”), wherein one degree Brix is 1gram of sucrose in 100 grams of solution. Degrees Brix represents thestrength of the solution as percentage by mass—if the solution containsdissolved solids other than pure sucrose, then the ° Bx onlyapproximates the dissolved solid content. The concentration of sucrosealso can be determined by density measurement, in the case of puresolutions. If a determination of both identity and concentration isneeded, a commercially available enzymatic kit can be applied todetermine concentration and discriminate sucrose from otherdisaccharides and carbohydrates. A sucrose assay kit, such as the onesold by Sigma-Aldrich (St. Louis, Mo.) as Catalog number SCA20 may beused.

In exemplary aspects, each sugar solution comprises sucrose. Inexemplary aspects, the method comprises loading into a rotor (e.g., azonal rotor) an AAV fraction (e.g., a solution comprising AAV) with atleast two sucrose solutions, each sucrose solution of which (a) has adifferent sucrose concentration and (b) comprises sucrose at aconcentration ranging from about 45% (w/w) to about 65% (w/w) sucrose.In certain embodiments, at least one solution comprises sucrose at aconcentration of between 52-58% (w/w) sucrose, and at least anothersolution comprises sucrose at a concentration of between 57-63% (w/w)sucrose, and optionally at least another solution comprises sucrose at aconcentration of between 47-53% (w/w) sucrose. In certain embodiments,at least one solution comprises sucrose at a concentration of between54-56% (w/w) sucrose, and at least another solution comprises sucrose ata concentration of between 59-61% (w/w) sucrose, and optionally, atleast another solution comprises sucrose at a concentration between49-51% (w/w) sucrose. In certain embodiments, at least one solutioncomprises sucrose at a concentration greater than about 54% (w/w)sucrose, and at least another solution comprises sucrose at aconcentration greater than about 59% (w/w) sucrose, and optionally, atleast another solution comprises sucrose at a concentration greater thanabout 49% (w/w) sucrose. In certain embodiments, at least one solutioncomprises sucrose at a concentration equal to or greater than about 55%(w/w) sucrose, at least one solution comprises sucrose at aconcentration equal to or greater than about 60% (w/w) sucrose, andoptionally, at least one solution comprises sucrose at a concentrationequal to or greater than about 50% (w/w) sucrose. In certainembodiments, at least one solution comprises sucrose at a concentrationequal of about 55% (w/w) sucrose, at least one solution comprisessucrose at a concentration of about 60% (w/w) sucrose, and optionally,at least one solution comprises sucrose at a concentration of about 50%(w/w) sucrose. In certain embodiments, the solution comprising AAV is abuffered solution (e.g., TrisHCl/NaCl buffer). In certain embodiments,the solution comprising AAV is a buffered ethylene glycol solution asdescribed above. In certain embodiments, the buffered ethylene glycolsolution is an aqueous solution. In certain embodiments, the bufferedethylene glycol solution is an aqueous solution that comprises 45-55%(w/w) of ethylene glycol and a buffer comprising TrisHCl and NaCl. Incertain embodiments, TrisHCl is at a concentration of about 20 to about50 mM. In certain embodiments, NaCl is at a concentration of about 100mM to about 750 mM or about 150 mM to about 500 mM. In certainembodiments, TrisHCl is at a concentration of about 20 to about 50 mMand NaCl is at a concentration of about 100 mM to about 750 mM, about150 mM to about 500 mM, or about 500 mM to about 750 mM. In certainembodiments, the pH of the solution comprising AAV is from 7.4 to 8.5,7.6 to 8.3, 7.8 to 8.5, 7.4 to 7.8, 7.6 to 8.0, 7.8 to 8.2, or 8.0 to8.5.

In exemplary aspects, the method comprises loading into a rotor (e.g., azonal rotor) an AAV fraction (e.g., a solution comprising AAV) with atleast two sucrose solutions, each sucrose solution of which (a) has adifferent sucrose concentration and (b) comprises sucrose at aconcentration ranging from about 50% (w/w) to about 60% (w/w) sucrose.In certain embodiments, at least one solution comprises sucrose at aconcentration of between 52-58% (w/w) sucrose and at least anothersolution comprises sucrose at a concentration of between 57-63% (w/w)sucrose. In certain embodiments, at least one solution comprises sucroseat a concentration of between 54-56% (w/w) sucrose and at least anothersolution comprises sucrose at a concentration of between 59-61% (w/w)sucrose. In certain embodiments, at least one solution comprises sucroseat a concentration greater than about 54% (w/w) sucrose and at leastanother solution comprises sucrose at a concentration greater than about59% (w/w) sucrose. In certain embodiments, at least one solutioncomprises sucrose at a concentration equal to or greater than about 55%(w/w) sucrose and at least one solution comprises sucrose at aconcentration equal to or greater than about 60% (w/w) sucrose. Incertain embodiments, at least one solution comprises sucrose at aconcentration equal of about 55% (w/w) sucrose and at least one solutioncomprises sucrose at a concentration of about 60% (w/w) sucrose. Incertain embodiments, the solution comprising AAV is a buffered solution(e.g., TrisHCl/NaCl buffer). In certain embodiments, the solutioncomprising AAV is a buffered ethylene glycol solution as describedabove.

In exemplary aspects, the method comprises loading into a rotor (e.g., azonal rotor) an AAV fraction (e.g., a solution comprising AAV) with atleast three sucrose solutions, each sucrose solution of which (a) has adifferent sucrose concentration and (b) comprises sucrose at aconcentration ranging from about 45% (w/w) to about 65% (w/w) sucrose,optionally ranging from about 50% (w/w) to about 60% (w/w). In certainembodiments, at least one solution comprises sucrose at a concentrationof between 47-53% (w/w) sucrose, at least another solution comprisessucrose at a concentration of between 52-58% (w/w) sucrose, and at leastanother solution comprises sucrose at a concentration of between 57-63%(w/w) sucrose. In certain embodiments, at least one solution comprisessucrose at a concentration of between 49-51% (w/w) sucrose, at leastanother solution comprises sucrose at a concentration of between 54-56%(w/w) sucrose, and at least another solution comprises sucrose at aconcentration between 59-61% (w/w) sucrose. In certain embodiments, atleast one solution comprises sucrose at a concentration greater thanabout 49% (w/w) sucrose, at least another solution comprises sucrose ata concentration greater than 54% (w/w) sucrose, and at least anothersolution comprises sucrose at a concentration greater than about 59%(w/w) sucrose. In certain embodiments, at least one solution comprisessucrose at a concentration equal to or greater than about 50% (w/w)sucrose, at least one solution comprises sucrose at a concentrationequal to or greater than about 55% (w/w) sucrose, and at least onesolution comprises sucrose at a concentration equal to or greater thanabout 60% (w/w) sucrose. In certain embodiments, at least one solutioncomprises sucrose at a concentration equal of about 50% (w/w) sucrose,at least one solution comprises sucrose at a concentration of about 55%(w/w) sucrose, and at least one solution comprises sucrose at aconcentration of about 60% (w/w) sucrose. In certain embodiments, thesolution comprising AAV is a buffered solution (e.g., TrisHCl/NaClbuffer). In certain embodiments, the solution comprising AAV is abuffered ethylene glycol solution as described above.

In exemplary aspects, the method comprises loading into the rotor (e.g.,zonal rotor) three, four, five, six, or more sucrose solutions, eachsucrose solution of which (a) has a different sucrose concentration and(b) comprises sucrose at a concentration ranging from about 45% (w/w) toabout 65% (w/w) sucrose, optionally ranging from about 50% (w/w) toabout 60% (w/w), or optionally ranging from about 55% (w/w) to about 60%(w/w). The complete ranges of sucrose concentrations are as listed abovewith respect to the ranges of sugar solutions. The ratio of sucrosesolutions to AAV fraction and/or other sugar solutions is as listedabove.

In exemplary aspects, the volume of the sucrose solution with the lowestsucrose concentration and the volume of the sucrose solution with thehighest sucrose concentration are about 20% to about 30% and about 20%to about 30% of the rotor volume, respectively. In certain embodiments,the volume of the sucrose solution with the lowest sucroseconcentration, the volume of the sucrose solution with the intermediatesucrose concentration and the volume of the sucrose solution with thehighest sucrose concentration are each about 25% of the rotor volume. Inexemplary aspects, the volume of the sucrose solution with the lowestsucrose concentration, the sucrose solution with the intermediatesucrose concentration and the sucrose solution with the highest sucroseconcentration are about 20% to about 30%, about 10% to about 15%, andabout 10% to about 15% of the rotor volume, respectively. In certainembodiments, the volume of the sucrose solution with the lowest sucroseconcentration, the sucrose solution with the intermediate sucroseconcentration and the sucrose solution with the highest sucroseconcentration are about 25%, about 12.5%, and about 12.5% of the rotorvolume, respectively. In certain embodiments, the ultracentrifugationcore volume or the rotor volume is within a range of about 200 mL toabout 10,000 mL, within a range of about 700 mL to about 8500 mL, orwithin a range of about 700 mL to about 7,700 m L.

A wide variety of ultracentrifugation cores are available and known tothose in the art. For example, the smallest ultracentrifugation coreavailable is the 200 mL Hitachi CC40S, while the largest core is the8,000 mL Hitachi CC40. The CC40CT3 Core E all other continuous flowultracentrifuges and cores from other suppliers may be used.

In exemplary aspects, the method comprises operating an ultracentrifugecomprising the rotor (e.g., zonal rotor) in batch mode, whereupon asugar gradient is formed. In certain embodiments, the ultracentrifuge isoperated at a first rotational speed of less than 10,000 rpm. In certainembodiments, the first rotational speed is about 3,000 rpm to about6,000 rpm (e.g., 4,000 rpm or 5,000 rpm) and the first rotation speed isachieved within about 15 to about 25 minutes. In certain embodiments,the ultracentrifuge is operated at a first rotational speed at atemperature between about 2° C. and about 10° C. In certain embodiments,the ultracentrifuge is operated at the first rotational speed for thepurpose of ultimately achieving a higher rotational speed. In certainembodiments, the ultracentrifuge is accelerated to a second rotationalspeed, which is at least 2× or at least 3× greater than the firstrotational speed. In certain embodiments, second rotational speed isachieved upon accelerating the ultracentrifuge for about 5 to about 60min. In certain embodiments, the ultracentrifuge is operated at a secondrotational speed greater than or about 30,000 rpm. In certainembodiments, the ultracentrifuge is operated at a second rotationalspeed greater than or about 30,000 rpm and less than or about 50,000rpm. In certain embodiments, the second rotational speed is betweenabout 30,000 rpm and about 40,000 rpm, e.g., about 35,000 rpm. Incertain embodiments, ultracentrifuge is operated at a second rotationalspeed for at least or about 3 hours, at least or about 4 hours, at leastor about 5 hours, or at least or about 6 hours, at least or about 10hours, at least or about 12 hours, at least or about 14 hours, at leastor about 16 hours, at least or about 18 hours, at least or about 20hours, or at least or about 22 hours. In certain embodiments,ultracentrifuge is operated at a second rotational speed for at least orabout 4 hours. In certain embodiments, ultracentrifuge is operated at asecond rotational speed for about 16 to about 20 hours. In certainembodiments, ultracentrifuge is operated at a second rotational speedfor at least or about 16 hours. In certain embodiments, theultracentrifuge is operated at a second rotational speed at atemperature between about 15° C. and about 30° C., e.g., at least about18° C., between about 20° C. and about 25° C. (e.g., about 22° C.). Incertain embodiments, the ultracentrifuge is subsequently operated todecelerate the rotational speed, e.g., to the first rotational speed,e.g., about 4,000 rpm, optionally, over the course of about 5 to about90 minutes. In certain embodiments, the deceleration occurs at atemperature less than about 18° C., between about 2° C. and about 10°C., or less than about 8° C. In certain embodiments, the ultracentrifugeis operated at a temperature less than about 8° C. at least 30 minutesbefore the ultracentrifuge is stopped.

In exemplary aspects, the method comprises obtaining a fraction of thesugar gradient to obtain an AAV fraction. In certain embodiments, theobtained AAV fraction has greater than 60% of full capsids. In certainembodiments, the obtained AAV fraction has 70-80% of full capsids. Incertain embodiments, one or more fractions of the sugar gradient areobtained, and, in certain embodiments, the fractions are obtained fromthe gradient containing higher density fractions. In certainembodiments, the fractions equivalent to fractions 7-11 as shown in FIG.8 are obtained. In certain embodiments, the fractions equivalent tofractions 10-14 as described in Example 9 are obtained. In certainembodiments, the fractions equivalent to fractions 1-8 as described inExample 16 are obtained. In certain embodiments, any fraction containingfull capsids from empty capsids are separated by their densitydifference as they elute from the rotor in the density gradient. Thecontrol of the separation can be achieved either by fractionating insmall volumes followed by analytical testing [e.g., as described inExample 9], or by fractionating according to UV signals from inlinemeasurement of UV signals (e.g. UV254 nm/UV280 nm signals and inlinecontrol of their ratio). In certain embodiments, the AAV fraction isobtained from the zone with higher sucrose density at stepultracentrifugation. In certain embodiments, the obtained AAV (e.g.,AAV8) fraction is confirmed as being mostly full capsids (e.g., >60%full capsids) by the ratio of [the UV signal at 254 nm]/[the UV signalat 280 nm] independent from the volume of the UC core used. If thisratio (OD 254 nm/OD 280 nm)>1, then the fraction is full capsids.

In exemplary aspects, the methods of the present disclosures yield anAAV product wherein at least 50% of the AAV capsids are full AAVcapsids. In certain embodiments, the methods of the present disclosuresyield an AAV product wherein at least 55% of the AAV capsids are fullAAV capsids. In certain embodiments, the methods of the presentdisclosures yield an AAV product wherein at least 60% of the AAV capsidsare full AAV capsids. In certain embodiments, the methods of the presentdisclosures yield an AAV product wherein at least 70% of the AAV capsidsare full AAV capsids. In certain embodiments, the methods of the presentdisclosures yield an AAV product wherein at least 80% of the AAV capsidsare full AAV capsids. In certain embodiments, the methods of the presentdisclosures yield an AAV product wherein at least 90% of the AAV capsidsare full AAV capsids. It will be appreciated that “at least 50%” refersto a range wherein 50% is the minimum percentage. The maximum percentageof such a range is, in various embodiments, 100%, although sub-rangesalso are contemplated herein where the maximum percentage is, e.g., 99%,98%, 95%, 80%, 85%, and the like. Suitable methods for measuring theamount of full capsids vs. empty capsids are known in the art andinclude, for example, negative stain transelectron microscopy andanalytic anion exchanger chromatography using a detection set-up thatallows discrimination between full and empty capsids. See, e.g., Lock etal., Human Gene Ther Part B 23:56-64 (2012); and Qu et al., J VirolMethods 140: 183-192 (2007).

In exemplary aspects, the sugar (e.g., sucrose) solutions differ inconcentration by about 5% (w/w) to about 15% (w/w). In certainembodiments, the sugar solutions differ in sucrose-equivalentconcentration by about 5% (w/w) to about 10% (w/w), about 4% (w/w) toabout 8% (w/w), or about 2% (w/w) to about 3% (w/w). In certainembodiments, the sugar solutions comprise sucrose and differ in sucroseconcentration by about 5% (w/w) to about 10% (w/w), about 4% (w/w) toabout 8% (w/w), or about 2% (w/w) to about 3% (w/w).

In exemplary aspects, the method of purifying recombinantadeno-associated virus (rAAV) particles and/or the method of separatingfull viral particles from the empty capsids comprises ultracentrifuginga fraction comprising rAAV particles with two sucrose solutions, eachsucrose solution comprising a different sucrose concentration, rangingfrom about 50% (w/w) to about 65% (w/w) at a first rotational speed ofless than 10,000 rpm (e.g., 4,000 rpm) for about 15 min to about 45 minor about 17 min to about 25 min, and at a second rotational speed withinthe range of about 30,000 to about 40,000 rpm (e.g., 35,000 rpm) for atleast 4 hours or about 16 to about 20 hours. In certain embodiments, onesolution comprises sucrose at a concentration equal of about 55% (w/w)sucrose and one solution comprises sucrose at a concentration of about60% (w/w) sucrose. In exemplary aspects, the ultracentrifugation step iscarried out as essentially described in Example 6.

In exemplary aspects, the method of purifying recombinantadeno-associated virus (rAAV) particles and/or the method of separatingfull viral particles from the empty capsids comprises ultracentrifuginga fraction comprising rAAV particles with three sucrose solutions, eachsucrose solution comprising a different sucrose concentration, rangingfrom about 45% (w/w) to about 65% (w/w), at a first rotational speed ofless than 10,000 rpm (e.g., 4,000 rpm) for about 15 min to about 45 minor about 17 min to about 25 min, and at a second rotational speed withinthe range of about 30,000 to about 40,000 rpm (e.g., 35,000 rpm) for atleast 4 hours or about 16 to about 20 hours. In certain embodiments, onesolution comprises sucrose at a concentration equal of about 50% (w/w)sucrose, one solution comprises sucrose at a concentration of about 55%(w/w) sucrose, and one solution comprises sucrose at a concentration ofabout 60% (w/w) sucrose. In exemplary aspects, the ultracentrifugationstep is carried out as essentially described in Examples 7 or 9.

Source of rAAV Particles

With regard to the methods of the present disclosure, the AAV may be ofany AAV serotype. In certain embodiments, the AAV purified by themethods described herein are of AAV1 serotype, AAV2 serotype, AAV3serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype,AAV8 serotype, AAV9 serotype, or AAV10 serotype. In certain embodiments,the AAV particles purified by the methods described herein are of AAV8serotype. With regard to the methods of the invention, the AAV fractionwhich is loaded into the rotor is in exemplary aspects a concentratedAAV fraction. In certain embodiments, the AAV fraction loaded into therotor comprises at least 1×10¹⁰, 1×10¹¹ or 1×10¹² AAV capsids per mL. Incertain embodiments, the AAV fraction loaded into the rotor comprises atleast 1×10¹² AAV capsids per mL, wherein the AAV capsids include emptyAAV capsids and full AAV capsids.

In certain embodiments, the AAV fraction represents an AAV fractionproduced by transfected host cells. In certain embodiments, the AAVfraction represents a supernatant harvested from a cell culturecomprising host cells transfected with a triple plasmid system, whereinone plasmid of the system comprises a gene or cDNA of interest, oneplasmid encodes capsid protein VP1, capsid protein VP2 and/or capsidprotein VP3. In certain embodiments, VP1, VP2, and/or VP3 are AAV8 VP1,AAV8 VP2, and/or AAV8 VP3. Triple plasmid transfection for purposes ofrAAV production is known in the art. See, e.g., Qu et al., 2015, supra,and Mizukami et al., “A Protocol for AAV vector production andpurification.” PhD dissertation, Division of Genetic Therapeutics,Center for Molecular Medicine, 1998; and Kotin et al., Hum Mol Genet20(R1): R2-R6 (2011). In certain embodiments, the transfection may becarried out using inorganic compounds, e.g., calcium phosphate, ororganic compounds, polyethyleneimine (PEI), or non-chemical means, e.g.,electroporation.

In certain embodiments, the host cells are adherent cells. In certainembodiments, the host cells are suspension cells. In certainembodiments, the host cells are HEK293 cells or Sf9 cells (e.g.,baculovirus infected Sf9 cells). In certain embodiments, the cellculture comprises culture medium which is serum and protein free. Incertain embodiments, the medium is chemically defined and is free ofanimal derived components, e.g., hydrolysates.

In certain embodiments, the fraction comprising rAAV particlesrepresents a fraction comprising HEK293 cells transfected with a tripleplasmid system. In certain embodiments, the fraction comprising rAAVparticles represents a fraction of a supernatant harvested about 3 toabout 5 days after transfection of the HEK293 cells or when the cellculture has a cell density of greater than or about 5×10⁶ cells/mL andhas a cell viability of greater than or about 50%. In certainembodiments, the fraction comprising rAAV particles represents afraction comprising HEK293 cells as described in Example 1.

In certain embodiments, the AAV is prepared by a triple plasmidtransfection followed by harvest from one to 5 days later. In certainembodiments, the AAV is prepared from cell disruption.

In certain embodiments, the AAV is prepared by the following: The HEK293cells are adherent and grown in a commercially-available culture mediumthat may be chemically-defined and may be free of animal-derivedcomponents, e.g. serum and proteins. The cells are cultured to a celldensity of about 3×10⁶ to about 12 cells/ml, e.g., about 6×10⁶ to about10 cells/ml. The cells are then split in about a 1:2 ratio such that thecell density is about 3-5×10⁶ cells/ml. After the split, the cells maybe transfected with three plasmids that include (1) a helper plasmidcapable of providing one or more helper viral functions essential AAVproduction, (2) a plasmid that encodes for one or more genes involved incapsid generation, replication and packaging of the virus, and (3) aplasmid comprising a gene of interest (GOI) to be packaged into theresulting rAAV particle. For example, the GOI may be a vector DNAcomprising human coagulation Factor IX Padua in a single strandedself-complementary form, with the vector DNA having a full length of 2.6kB. As another example, the GOI may be a vector DNA comprising humancoagulation Factor IX Padua in a double stranded self-complementaryform, with the vector DNA having a full length of 4.8 kB. As anotherexample, the GOI may be a vector DNA comprising a B-domain deleted humancoagulation Factor VIII in a single stranded self-complementary form,with the vector DNA having a full length of 4.8 kB. Other GOI may beused. Transfection may be carried out in a transient manner, such as byusing cationic polymers. Before elution, the HEK293 cell line may becultivated for at least about 3 days, e.g., 3-5 days, before harvesting.

Additional Steps

The methods of the present disclosure comprise any combination of stepsdisclosed herein, and may optionally be combined with one or moreadditional steps. Accordingly, in exemplary aspects, the methods of thepresent disclosure further comprise the step of transfecting host cellswith a triple plasmid system as described herein. In exemplary aspects,the methods of the present disclosure comprise harvesting a supernatantfrom a cell culture comprising host cells, e.g., HEK293 cells or Sf9(e.g., baculovirus infected Sf9 cells), transfected with a tripleplasmid system. In exemplary aspects, the methods of the presentdisclosure comprise harvesting the supernatant about 3 to about 5 daysafter transfection of the HEK293 cells or when the cell culture has acell density of greater than or about 5×10⁶ cells/mL and has a cellviability of greater than 50%. In certain embodiments, the AAV isprepared from cell disruption. In exemplary aspects, the transfectionand harvesting step occurs prior to the ultracentrifugation stepdescribed herein. The methods of the present disclosure may comprise yetother additional steps, which may further increase the purity of the AAVand remove other unwanted components and/or concentrate the fractionand/or condition the fraction for a subsequent step. The additionalsteps may occur before or after the ultracentrifugation step describedabove.

In exemplary aspects, the method comprises a depth filtration step. Inexemplary aspects, the method comprises subjecting a fraction of atransfected HEK293 cell culture supernatant to depth filtration using afilter comprising cellulose and perlites and having a minimumpermeability of about 500 L/m². In exemplary aspects, the method furthercomprises use of a filter having a minimum pore size of about 0.2 μm. Inexemplary aspects, the depth filtration is followed by filtrationthrough the filter having a minimum pore size of about 0.2 μm. Inexemplary aspects, one or both of the depth filter and filter having aminimum pore size of about 0.2 μm are washed and the washes arecollected. In exemplary aspects, the washes are pooled together andcombined with the filtrate obtained upon depth filtration and filtrationwith the filter having a minimum pore size of about 0.2 μm. Example 2provides an exemplary method of depth filtration and filtration througha filter having a minimum pore size of about 0.2 μm. In exemplaryaspects, the depth filtration step and other filtration step occursprior to the ultracentrifugation step described herein.

In certain embodiments, harvesting is conducted by the following: Thesupernatant of the cell culture is harvested at this time via depthfiltration, e.g., by filtering about 150 to about 250 L ofAAV-containing cell suspension through a depth filter element and apolyethersulfone element. The flow rate can be from about 40 kg/hour toabout 280 kg/hour, or from about 60 kg/hour to 240 kg/hour. The totalpressure is less than 1.7 bar. The two filters are flushed with about 10to about 30 L of Tris Buffered Saline (TBS) buffer. The filteredfermentation broth, or harvest, is then collected. The depth filtrationprocedure can remove at least 70% of the protein and 60% of HEK293 cellDNA, while retaining at least 60% of 65% of the AAV particles.

In certain embodiments, the harvest containing the AAV fraction issubjected to ultrafiltration/diafiltration (UF/DF) and TFF toconcentrate and condition the harvest as follows: The harvest isconcentrated about 10-fold to about 20-fold to a target volume of about10 L to about 15 L using TFF. The concentrated harvest is then subjectedto two diafiltration steps to condition the concentrated harvest to a pHof about 8.3 to about 8.7 and a conductivity of about 13 mS/cm to about17 mS/cm. Each diafiltration step may be performed via a 5-fold volumeexchange with a diafiltration buffer, e.g., the first buffer comprises50 mM TRIS and 500 mM NaCl, having a pH of 8.5±0.2, and at 25° C., andthe second buffer comprises 50 mM TRIS and 125 mM NaCl, having a pH of8.5±0.2, and at 25° C. TFF is then undertaken to concentrate theretentate to a final target volume of about 8 L to about 12 L. Theconcentrate is then filtered through a 0.2 μm filter element. The AAVyield may increase by flushing the filter element with about 1.5 toabout 2.5 of the second diafiltration buffer.

In exemplary aspects, the methods of the present disclosure comprise oneor more chromatography steps. In exemplary aspects, the methods comprisea negative chromatography step whereby unwanted components bind to thechromatography resin and the desired AAV does not bind to thechromatography resin. In exemplary aspects, the methods comprise anegative anion exchange (AEX) chromatography step, or an AEXchromatography step in the “non-binding mode”. Example 4 describes sucha step. Accordingly, in exemplary embodiments, the methods of purifyingAAV particles comprise performing negative anion exchange (AEX)chromatography on a fraction comprising AAV particles by applying thefraction to an AEX chromatography column or membrane under conditionsthat allow for the AAV to flow through the AEX chromatography column ormembrane and collecting AAV particles. In exemplary aspects, thefraction is applied to the AEX chromatography column or membrane with aloading buffer comprising about 100 mM to about 150 mM salt, e.g., NaCl,optionally, wherein the pH of the loading buffer is about 8 to about 9.In exemplary aspects, the loading buffer comprises about 115 mM to about130 mM salt, e.g., NaCl, optionally, wherein the loading buffercomprises about 120 mM to about 125 mM salt, e.g., NaCl. In exemplaryaspects, the negative AEX step occurs prior to the ultracentrifugationstep described herein.

In exemplary aspects, the methods of the present disclosure compriseconcentrating an AAV fraction using an ultra/diafiltration system. Inexemplary aspects, the methods of the present disclosure comprise onemore tangential flow filtration (TFF) steps. In exemplary aspects, theAAV fraction undergoes ultra-/dia-filtration. In exemplary aspects, theAAV fraction is concentrated with the ultra/diafiltration system beforea step comprising performing negative AEX chromatography, after a stepcomprising performing negative AEX chromatography, or before and aftercomprising performing negative AEX chromatography. Examples 3 and 5describe such TFF steps. In exemplary aspects, the TFF steps occur priorto the ultracentrifugation step described herein.

In certain embodiments, an additional TFF step is performed afternegative AEX chromatography as follows: The AAV-containing flow throughfraction obtained from AEX chromatography is concentrated anddiafiltered against a buffer, e.g. one comprising about 500 mM NaCl,about 50 mM TrisHCl; at pH of about 8.3 to about 8.7, to condition theproduct for ultracentrifugation. A TFF step is then carried out.

In certain embodiments, empty and full AAV particles are separated fromone another by “two sucrose protocol” ultracentrifugation as follows. Aconcentrate comprising AAV in a TrisHCl/NaCl buffer followed by a firstsucrose solution comprising sucrose in a 55% (w/w) sucrose concentrationand then a second sucrose solution comprising sucrose in a 60% (w/w)sucrose concentration. In certain embodiments, the AAV solutioncomprises about 50 mM TrisHCl, and about 500 mM NaCl. In certainembodiments, the sucrose solution comprises about 50 mM TrisHCl andabout 136 mM NaCl. Ultracentrifugation is initially conducted at about4,000 rpm at a temperature of 2-10° C., with the speed increased toabout 33,000 rpm to about 37,000 rpm, the temperature increased to about20° C. to about 25° C., and then held for about 4 hours, about 14 hoursto about 20 hours, or from about 16 hours to about 20 hours. Fractionsare then collected.

In certain embodiments, empty and full AAV particles are separated fromone another by “two sugar protocol” ultracentrifugation as follows. Aconcentrate comprising AAV and a buffered ethylene glycol solutioncontaining 45-50% (w/w) ethylene glycol in a TrisHCl/NaCl bufferfollowed by a first sucrose solution comprising sucrose in a 55% (w/w)sucrose concentration and then a second sucrose solution comprisingsucrose in a 60% (w/w) sucrose concentration. In certain embodiments,the AAV solution comprises about 55% ethylene glycol, about 50 mMTrisHCl, and about 750 mM NaCl. In certain embodiments, the sucrosesolution comprises about 50 mM TrisHCl and about 136 mM NaCl.Ultracentrifugation is initially conducted at about 4,000 rpm at atemperature of 2-10° C., with the speed increased to about 33,000 rpm toabout 37,000 rpm, the temperature increased to about 20° C. to about 25°C., and then held for about 4 hours, about 14 hours to about 20 hours,or from about 16 hours to about 20 hours. Fractions are then collected.

In certain embodiments, empty and full AAV particles are separated fromone another by “three sugar protocol” ultracentrifugation as follows. Aconcentrate comprising AAV in a TrisHCl/NaCl buffer followed by a firstsucrose solution comprising sucrose in a 50% (w/w) sucroseconcentration, a second sucrose solution comprising sucrose in a 55%(w/w) sucrose concentration, and a third sucrose solution comprisingsucrose in a 60% (w/w) sucrose concentration. The first sucrose solutionis loaded into the bottom of the rotor; followed by the second sucrosesolution and the third sucrose solution. In certain embodiments, the AAVsolution comprises about 50 mM TrisHCl, and about 500 mM NaCl. Incertain embodiments, the sucrose solution comprises about 50 mM TrisHCland about 136 mM NaCl. Ultracentrifugation is initially conducted atabout 4,000 rpm at a temperature of 2-10° C., with the speed increasedto about 33,000 rpm to about 37,000 rpm, the temperature increased toabout 20° C. to about 25° C., and then held for about 3 hours to about 6hours, from about 4 hours, about 14 hours to about 20 hours, or fromabout 16 hours to about 20 hours. Fractions are then collected.

In certain embodiments, empty and full AAV particles are separated fromone another by “three sucrose protocol” ultracentrifugation as follows.A concentrate comprising AAV and a buffered ethylene glycol solutioncontaining 45-50% (w/w) ethylene glycol in a TrisHCl/NaCl bufferfollowed by a first sucrose solution comprising sucrose in a 50% (w/w)sucrose concentration, a second sucrose solution comprising sucrose in a55% (w/w) sucrose concentration, and a third sucrose solution comprisingsucrose in a 60% (w/w) sucrose concentration. The first sucrose solutionis loaded into the bottom of the rotor; followed by the second sucrosesolution and the third sucrose solution. In certain embodiments, the AAVsolution comprises about 55% ethylene glycol, about 50 mM TrisHCl, andabout 750 mM NaCl. In certain embodiments, the sucrose solutioncomprises about 50 mM TrisHCl and about 136 mM NaCl. Ultracentrifugationis initially conducted at about 4,000 rpm at a temperature of 2-10° C.,with the speed increased to about 33,000 rpm to about 37,000 rpm, thetemperature increased to about 20° C. to about 25° C., and then held forabout 3 hours to about 6 hours, from about 4 hours, about 14 hours toabout 20 hours, or from about 16 hours to about 20 hours. Fractions arethen collected.

In exemplary aspects, the methods of the present disclosure comprisetreating a fraction comprising AAV particles with a solvent detergent toinactivate lipid enveloped viruses. Example 8 describes an exemplarymethod of lipid enveloped virus inactivation. In exemplary aspects, thesolvent detergent treatment step occurs after the ultracentrifugationstep described herein.

In exemplary aspects, the methods of the present disclosure comprisefiltration of a fraction comprising rAAV particles to remove viruses ofgreater size than the rAAV particles in the fraction. In exemplaryaspects, the method of the present disclosure comprises filtration of afraction comprising AAV to remove viruses sized greater than or about 35nm. In exemplary aspects, the pore size of the filter is in thenanometer range, and, in exemplary aspects, the method comprisesnanofiltration. In exemplary aspects, the method of the presentdisclosure comprises use of a nanofilter of pore size in the range of 35nanometer±2 nanometer, as determined by a water flow method. Anexemplary nanofilter having such pore size contains bundles ofmicro-porous hollow-fibers constructed of a natural hydrophiliccuprammonium regenerated cellulose. Classification of the type of filteris dependent on membrane structure, material, and vendor. In exemplaryaspects, the nanofilter is tested with an integrity leakage test toconfirm that the filter is free from pinholes or large defects andpre-washed with formulation buffer.

An exemplary nanofiltration is described herein as Example 10.

In exemplary aspects, the fraction comprising rAAV particles, e.g., ananion exchange eluate, is pre-diluted with elution buffer (PBS+600 mMNaCl) to adjust concentration of the virus particles.

In exemplary aspects, during the filtration step, a pressure differenceover the filter is maintained. In exemplary aspects, the pressure(pressure drop across the filter) is about 0.02 MPa to about 0.1 MPa. Inexemplary aspects, the pressure (e.g., pressure drop across the filter)is about 0.02 MPa to about 0.08 MPa. In case the filter is run indead-end mode, the pressure difference can be effected by the feedpressure of the sample applied (i.e., by adjustment of a pump to aspecific flow, which affects the feed pressure). In exemplary aspects,the filtration is conducted under constant pressure not exceeding 0.1MPa in a dead-end mode through the nanofilter. In exemplary aspects, thefilter is run in a tangential flow method. In exemplary aspects, theyield of recovered virus particles is increased by post-washing thenanofilter with buffer (e.g. formulation buffer). In exemplary aspects,post-integrity testing of the nanofilter is performed with a leakagetest and/or a gold particle test to determine that the nanofilter poresize distribution does not change and that the nanofilter retains itsintegrity during filtration. In exemplary aspects, more than 50 L ofsolution is applied per m² filter area to increase the sample yield.

In exemplary aspects, the filtration step for removal of viruses largerthan the rAAV particles occurs once during the process of the presentdisclosure. In exemplary aspects, the filtration step occurs twiceduring the process. In exemplary aspects, the filtration step forremoval of viruses larger than the rAAV particles occurs after theultracentrifugation step described herein. In exemplary aspects, thefiltration step for removal of viruses larger than the rAAV particlesoccurs after a polish step.

In exemplary aspects, the methods of the present disclosure comprise apolish step comprising performing AEX chromatography, optionally with acolumn comprising tentacle gel. Example 8 describes an exemplary methodcomprising such a polish step. In exemplary aspects, the polish stepoccurs after the ultracentrifugation step described herein.

In exemplary aspects, the methods of the present disclosure comprise oneor more quality control steps, e.g., steps to measure the potency.DNA/AAV ratio, or specific activity of the AAV fractions obtained afterone or more steps (e.g., after each step) of the process. In exemplaryaspects, the methods of the present disclosure comprise assaying for thepresence of AAV in fractions by using qPCR, e.g., ITR qPCR. ITR qPCR isa quantitative PCR based assay to measure the amount of AAV InvertedTerminal Repeat (ITR) nucleic acid in the fraction. Other AAV-specific,or AAV8-specific sequences can be assayed by qPCR.

In exemplary aspects, the methods of the present disclosure compriseassaying for the presence of AAV in fractions by using ELISA. Inexemplary aspects, the ELISA is a sandwich ELISA. In exemplary aspects,the sandwich ELISA comprises an antibody specific for an AAV epitope. Inexemplary aspects, the AAV epitope is a conformational epitope presenton assembled AAV capsids. A suitable method of testing DNA/AAV ratio isdescribed herein as Example 12. As discussed herein, the ELISA mayreplace qPCR as a way to determine potency of an AAV fraction. Inexemplary aspects, the methods of the present disclosure comprisetesting an AAV fraction via an AAV-specific ELISA and the methods do notinclude a method of measuring potency via quantitative PCR. In exemplaryaspects, the AAV-specific ELISA is sufficient to provide arepresentative reading on potency of the AAV fraction, because themajority of the capsids in the AAV fraction are full capsids.

In exemplary aspects, the methods of the present disclosure comprise anELISA specific for AAV after one or more of the steps of the presentdisclosure. In exemplary aspects, the methods of the present disclosurecomprise testing an AAV fraction obtained after ultracentrifugation viaan AAV-specific ELISA to determine the DNA/AAV ratio of the AAV in thatfraction. In exemplary aspects, the methods of the present disclosurecomprise testing an AAV fraction obtained after depth filtration via anAAV-specific ELISA to determine the DNA/AAV ratio of the AAV in thatfraction. In exemplary aspects, the methods of the present disclosurecomprise testing an AAV fraction obtained after concentrating an AAVfraction using an ultra-/diafiltration system via an AAV-specific ELISAto determine the DNA/AAV ratio of the AAV in that fraction. In exemplaryaspects, the methods of the present disclosure comprise testing an AAVfraction obtained after a tangential flow filtration (TFF) step via anAAV-specific ELISA to determine the DNA/AAV ratio of the AAV in thatfraction. In exemplary aspects, the methods of the present disclosurecomprise testing an AAV fraction obtained after negative anion exchange(AEX) chromatography via an AAV-specific ELISA to determine the DNA/AAVratio of the AAV in that fraction. In exemplary aspects, the methods ofthe present disclosure comprise testing an AAV fraction obtained after apolish step via an AAV-specific ELISA to determine the DNA/AAV ratio ofthe AAV in that fraction.

In exemplary aspects, the method of the present disclosure comprises oneor a combination of steps illustrated in FIG. 1. In exemplary aspects,the method of the present disclosure comprises all steps illustrated inFIG. 1.

An AAV product produced by a method of the present disclosures isfurther provided herein. In exemplary aspects, the AAV product comprisesat least about 10¹² virus particles (vp) produced from about 1000 L ofstarting material (e.g., cell culture) or at least about 10¹³ virusparticles (vp) produced from about 1000 L of starting material (e.g.,cell culture) and wherein at least 50% or at least 55% of the AAVcapsids present in the AAV product are full AAV capsids. In exemplaryaspects, the AAV product of the present disclosures is highly pure,highly potent and suitable for clinical use in humans. In exemplaryaspects, the AAV product comprises AAV particles of a homogenouspopulation and high purity. In exemplary aspects, the AAV productcomprises full-length vector DNA. In exemplary embodiments, the AAVproduct is substantially free of unwanted contaminants, including butnot limited to, AAV particles containing truncated or incomplete vectorDNA, AAV particles with incomplete protein composition and oligomerizedstructures, or contaminating viruses, e.g., non AAV, lipid envelopedviruses. In exemplary embodiments, the AAV product contains a highamount of encoding cDNA of the protein of interest. In exemplaryaspects, the AAV product of the present disclosure is suitable foradministration to a human. In exemplary aspects, the AAV product issterile and/or of good manufacturing practice (GMP) grade. In exemplaryaspects, the AAV product conforms to the requirements set forth in theU.S. Pharmacopeia Chapter 1046 or the European Pharmacopoeia on genetherapy medicinal products or as mandated by the U.S. Food and DrugAdministration (USFDA) or the European Medicines Agency (EMA). Inexemplary aspects, the AAV product is a ready-to-use product for directadministration to a human with little to no processing or handling.

The following examples are given merely to illustrate the presentinvention and not in any way to limit its scope.

EXAMPLES Example 1

The following example describes an exemplary method of transfecting aHEK293 cell line with a triple plasmid system to produce rAAV particlescomprising a nucleic acid encoding a protein of interest.

Adherent HEK293 cells are grown in suspension conditions in acommercially-available culture medium that is chemically-defined andfree of animal-derived components, protein and serum. The cells arecultured to a cell density of approximately 6-10×10⁶ cells/ml. Beforetransfection a split of about 1:2 is performed to reach a cell densityof approximately 3-5×10⁶ cells/ml.

The cells are transfected with three plasmids: (1) a helper plasmid,which provides helper viral functions essential for a productive AAVinfection, (2) the repcap-plasmid, which carries all informationregarding capsid generation, replication and packaging of the virus, and(3) a plasmid containing the gene of interest (GOD, which is packagedinto the resulting rAAV particle. For example, the GOI can be a FactorIX Padua vector DNA (single stranded (˜2.6 kb) or double stranded (˜4.8kb)) or Factor VIII single stranded vector DNA (˜4.8 kb).

Transient transfection of the HEK293 cells is carried out using thecationic polymer, polyethylenimine (PEI). Briefly, PEI and plasmid DNAare incubated for more than 10 minutes at temperatures between 4 to 37°C. prior to blending the transfection mix with the cell culture, inorder for pre-complex formation of PEI and plasmid DNA to take place.Pre-formed complexes are added to the cell culture along with thetransfection mix for and incubated for about 3 to 4 hours at atemperature between 30° C. and 40° C., e.g., about 37° C. Thetransfection is stopped by adding a known stop medium comprising, forexample, a medium, e.g., Hyclone™ CDM4HEK293, a chemically defined,animal component free and protein-free cell culture medium, including0.6 g/L glutamine, into the transfected culture.

The rAAV particles carrying the GOI are in the HEK293 cell line over aperiod of 3-5 days post-transfection. As shown in FIGS. 2 and 3, theHEK293 cell culture exhibits high cell densities (e.g., greater thanabout 5×10⁶ cells/mL) with a viability of >50% at a time that is about 5days post-transfection.

Example 2

The following example describes an exemplary method of harvesting thesupernatant of a transfected HEK293 cell culture.

As discussed in Example 1, rAAV production occurs within the first 3 toabout 5 days after transfection. At the end of this timeframe, theHEK293 cell culture exhibits high cell densities (e.g., greater thanabout 5×10⁶ cells/mL) with a viability of >50%. The supernatant of thecell culture was harvested at this time via depth filtration. The AAVparticles were separated from cells and cell debris by filtering about200 L (±20 L) AAV-containing cell suspension through a cellulose-baseddepth filter element (e.g. Pall, STAX K900P; 4 m²) and a 0.2 μmpolyethersulfon (PES) filter element (e.g. Pall, ECV; 2×5 inch, inparallel). The typical flow rate through the filters was 160±100 kg/hourand the total pressure was <1.7 bar. The two filters were flushed withabout 10-30 L Tris Buffered Saline (TBS) buffer (20 mM Tris, 8 g/L NaCl,pH 7.4±0.2 at 25° C.). Together with the filter flush, the filteredfermentation broth is collected in a 500 L bag and is referred to as aharvest. Harvesting step parameters and characterization of the harvest(yield) after the harvesting step are found in Tables 1 and 2,respectively. FIG. 4 provides a continuous recording of pressure andflow rate of the harvest step.

TABLE 1 Harvest Step Parameters Process Stage Parameter/Material ValueBioreactor Temperature for harvest start 19-37° C. conditions Workingvolume start filtration 200 +/− 20 L during harvest Depth Membrane typePall, STAX K900P filter Membrane material Cellulose based Area depthfilter of K900 STAX 4 (=2 * 2) m² Pre rinse volume (VE-water) >50 L/m²Container between No, stax and ecv online STAX and ECV 0.2 μm Filtertype Pall, Supor ECV filter Filter material PES (Polyethersulfon) Poresize 0.2 μm Number of filter elements 2 * 5 inch Area 0.2 um filter(ECV; 5 inch) 1.04 m² Filtration Typical flow rate 160 +/− 100 kg/hourparameters Total pressure (depth + 0.2 μm) <1.7 bar Filter flush TBSbuffer filter flush volume 10-30 L (depth + 0.2 μm) Collection offiltrate and flush Pall, 200 L Allegro Mixer

TABLE 2 YIELD CHARACTERISTICS Protein by HEK293 HCP Bradford ITR-qPCRAAV ELISA HEK DNA ELISA Volume Turbidity Total Total Total Total TotalSample code: [ml] [NTU] [μg/ml] [mg] [vg/ml] (vg) [μg/ml] [mg] [ng/ml][μg] [μg/ml] [mg] Load 200.000 n.d. 597.5 119500- 1.09E+12 2.18E+17-3.65 730- 800.0 160000- 69.2 13840- STX_1 — 0.160 43 — 1.33E+11 — 1.294— <2.5 — 21.7 — STX_2 — 1.450 79.34 — 3.95E+11 — 2.505 — 119.0 — 45.2 —STX_3 — 2.250 211 — 5.64E+11 — 3.087 — 592.0 — 44.9 — STX_F — 3.480 164— 4.17E+11 — 1.479 — 512.0 — 24.6 — ECV_1 — 0.170 25 — 1.26E+11 — 0.921— <2.5 — 16.5 — ECV_2 — 0.170 79 — 3.01E+11 — 1.867 — 33.0 — 31.4 —ECV_3 — 0.520 77 — 4.01E+11 — 2.610 — 221.0 — 42.3 — ECV_4 — 0.480 86 —3.82E+11 — 2.862 — 280.0 — 42.8 — ECV_5 — 0.850 198 — 5.09E+11 — 2.731 —273.0 — 41.9 — ECV_6 — 1.670 251 — 6.14E+11 — 3.526 — 784.0 — 45.2 —ECV_P 201.800 2.530 169  34052 5.24E+11 1.06E+17  2.404 485  295.0 59531 38.3  7729 Sample Code Description Load Supernatant at 3 dayafter transfection (Time of harvest) STX_1 After depth filtration of 1/6of the harvest STX_2 After depth filtration of 1/2 of the harvest STX_3After depth filtration of 5/6 of the harvest STX_F After flushing depthfilter with TBS buffer ECV_1 After 0.2 μm filtration of 1/12 of theharvest ECV_2 After 0.2 μm filtration of 1/4 of the harvest ECV_3 After0.2 μm filtration of 5/12 of the harvest ECV_4 After 0.2 μm filtrationof 7/12 of the harvest ECV_5 After 0.2 μm filtration of 3/4 of theharvest ECV_6 After 0.2 μm filtration of 11/12 of the harvest ECV_P Poolof harvest and flush

As shown above in Table 2, over 70% of the protein and more than 62% ofthe HEK DNA found in the cell suspension prior to filtration was removedvia this filtration step. The amount of AAV Inverted Terminal Repeat(ITR) nucleic acid in the pooled fraction containing the harvest and thefilter flush (P) as measured by quantitative PCR (qPCR) was about halfof that found in the cell suspension prior to filtration. Also, asmeasured by ELISA, greater than 65% of the AAV of the pre-filtrationcell suspension was retained upon filtration.

Example 3

The following example describes an exemplary way of concentrating andconditioning a harvest via an ultrafiltration/diafiltration (UF/DF)tangential flow filtration (TFF) system.

The harvest obtained in Example 2 was subjected to an UF/DF TFF systemto concentrate and condition the harvest. In a first step, the harvestwas concentrated approximately 16-fold to a target volume of 12 L by TFFusing 3×0.5 m² Pall T-series Omega Centramate membranes 100 kDa (PartNo.: OS100T06). The concentrated harvest was then subjected to twodiafiltration steps to reduce media components and to condition theconcentrated harvest to a pH of 8.5 and a conductivity of 15 mS/cm. Thefirst diafiltration step was performed via a 5-fold volume exchange withDiafiltration Buffer 1 (DB1: 50 mM TRIS/500 mM NaCl/pH 8.5±0.2 at 25°C.). The second diafiltration step was conducted via a 5-fold volumeexchange with Diafiltration Buffer 2 (DB2: 50 mM TRIS/125 mM NaCl/pH8.5±0.2 at 25° C.). Each diafiltration step was carried out by using3×0.5 m² Pall T-series Omega Centramate membranes 100 kDa (Part No.:OS100T06). After the second diafiltration step, the retentate wasconcentrated to a final target volume of 10 L by TFF as described above.The 10 L of concentrate was then filtered through a 0.2 μm filterelement (5 inch, Pall ECV filter, Part No.: NP5LUECVP1S). In order toincrease AAV8 yield, the UF/DF membranes, the UF/DF system and the 0.2μm filter were flushed with approximately 2 L of DB2. The retentate andthe filter flush were collected and mixed in a bag in order to prepareit for the subsequent capture step. FIG. 7 shows the intermediate stepsof the TFF in silver stain (left) and Western blot (right). Parametersof the UF/DF TFF steps and characterization of the resulting conditionedconcentrate [post UF/DF TFF steps] are found in Tables 3 and 4,respectively.

Description of Silver Stain

NuPAGE 4-12% Bis-Tris Midi Gel 1.0 mm. 20 well Cat. Nr. WG1402BX10MES SDS Running Buffer. Invitrogen. Cat. Nr. NP0002SB+DTT Incubation 10 min bei 70° C. 10 min cool down JAA treatment

Description of Western Blot

NuPAGE 4-12% Bis-Tris Midi Gel 1.0 mm. 20 well Cat. Nr. WG1402BX10MES SDS Running Buffer. Invitrogen. Cat. Nr. NP0002SB+DTT Incubation 10 min bei 70° C. 10 min cool down JAA treatment

1st Antibody: Mab to VP1. VP2 and VP3 of AAV (Adeno-Associated Virus)

Protein A affinity chromatography

PROGEN61058

2nd Antibody: GOAT anti Mouse ALP

SIGMA A4656 1:2000 1 h

TABLE 3 Pressure [bar] TMP (trans- membrane Flux [l/h] Conductivity StepBuffer Volume Feed Retentate Filtrate pressure) Retentate Filtrate[mS/cm] Conditioning and Ultradiafiltration approx. 12 L 0.7-1.1 0.0-0.30.0-0.2 0.20-0.40 n.a. n.a. n.a. Equilibration buffer Ultrafiltrationn.a. to 12 L 1.4-1.8 0.9-1.4 0.1-0.6 1.00-1.09 240-420 110-260 11.75(1st Concentration) 1st Ultradiafiltration 5 Volume 1.4-1.5 0.8-1.00.1-0.2 1.00-1.06 401-425  92-118 48.1  Ultradiafiltration bufferchanges 2nd Ultradiafiltration 5 Volume 1.4 0.8 0.1 0.99-1.02 439-44782-94 15.57 Ultradiafiltration buffer changes Ultrafiltration n.a. to 10L 1.4 0.8 0.1 0.99-1.02 n.a. n.a. n.a. (2nd Concentration) Membraneflush Ultradiafiltration ~2 L 1.0 n.a. n.a. n.a. n.a. n.a. n.a. buffern.a. . . . not applicable

TABLE 4 Protein by Bradford ITR-qPCR AAV ELISA HEK293 HCP ELISA VolumeTotal Yield Total Yield Total Yield Total Yield Sample code: [ml][μg/ml] [mg] (%) [vg/ml] (vg) (%) [μg/ml] [mg] (%) [μg/ml] [mg] (%) Load201800 149.9 30247.8 100.0 4.12E+11 8.31E+16 100.0 2.2 449.2 100.0  39.4 7950.9 100.0  Retentate 12000 1672.3 20067.0 66.3 5.64E+126.77E+16 81.4 34.3 411.4 91.6 368.0 4416.0 55.5 Filtrate 189800 47.59017.4 31.7 7.97E+08 1.51E+14 0.2 <0.03125 — — n.d. — — Diaretentate 112000 1433.7 17203.8 56.9 5.25E+12 6.30E+16 75.8 35.1 420.8 93.7 271.03252.0 40.9 Diafiltrate 1 60000 31.0 1857 6 6.18E+08 3.71E+13 0.0<0.03125 — — n.d. — — Diaretentate 2 12000 1621.4 19456.4 64.3 5.20E+126.24E+16 75.1 42.8 513.3 114.3  284.0 3408.0 42.9 Diafiltrate 2 6000013.4 802 3 8.88E+08 5.33E+13 0.1 <0.03125 — — n.d. — — Retentate 118571202.0 14252.6 47.1 4.81E+12 5.70E+16 68.6 28.0 332.5 74.0 227.0 2691.533.9 (2 × diafiltrated) pooled with flush Post 0.2 μm filtration n.d. .. . not determined

As shown in Table 4, the volume of the resulting conditioned concentratewas about 6% of the initial volume, yet the percentage of AAV in theconcentrate was 74%, as measured by ELISA.

Example 4

This example demonstrates an exemplary method of negative chromatographywith an anion exchanger.

After concentration and diafiltration of the harvest by TFF as describedin Example 3, the AAV containing buffer is conditioned to 125 mM NaCl/50mM TrisHCl/pH 8.5 for negative chromatography using a Mustang anionexchanger membrane (Pall Part Number XT5000MSTGQP1). At this condition,the AAV does not bind onto the anion exchanger membrane and, instead,flows through the column. After loading the column, two total volumes(equal to 2× the membrane capsule) of a flush buffer (125 mM NaCl/50 mMTris HCl; pH 8.5) were used to flush any amount of nonbinding AAV8 outof the column. FIG. 5 provides the chromatogram of the flow throughproduct of an AEX Mustang Q chromatography in negative (non-binding)mode. An elution was carried out to examine the bound proteins. Table 5details the scheme of the negative chromatography step and Table 6details the yield.

TABLE 5 Step Buffer Inlet Flowrate CV Col. Pos. Outlet FractionEquilibration 1 0.5M NaOH B2 400 ml/min 5 1 down F1 Waste 0.5M NaOH 2Bypass Equilibration 2 TWA-Buffer A4 400 ml/min 5 1 down F1 Waste 2MNaOH 2 Bypass Equilibration 3 Equilibration A1 400 ml/min 4 1 down F1Waste buffer 2 Bypass Load with air sensor Sample-Load: S1 400 ml/min —1 down 0-0.59CV F1 Waste 2 Bypass Ab0.59 CV F2 FT Wash Equilibration A1400 ml/min 3 1 down 0-1.68CV F2 FT buffer 2 Bypass 1.68-3CV F1 WasteElution Elution buffer B1 400 ml/min 2.6 1 down 0-0.4CV F1 Waste E 2Bypass 0.4-2.6CV F3 Equilibration buffer: 50 mM Tris; 125 mM NaCl (pH8.5 ± 0.1 bei 25° C. Elution buffer: 20 mM Tris; 1000 mM NaCl (pH 9.0 ±0.1 bei 25° C.

TABLE 6 Volume ITR qPCR AAV8 ELISA AKT Code (g) vg/ml Vg % μg/ml μg %vg/μg L 11628.40 2.91E+12 3.38E+16 100 34.361 399563.45 100 8.47E+10 FT22054.20 7.45E+11 1.64E+16 48.56 11.782 259842.58 65.03 6.32E+10 E21009.20 8.31E+10 1.75E+15 5.16 1.055 22164.71 5.55 7.88E+10 Protein byBradford In vitro OR-1300443 HEK293 HCP ELISA Code biopotency μg/ml mg %μg/ml mg % L 1.54 1295.98 15070.17 100.00 269 3128.04 100.00 FT 1.64234.55 5172.81 34.32 107 2359.80 75.44 E — 108.04 2269.83 15.06 — — — L= Loaded fraction; FT = flow through fraction; E = eluted fraction

As shown in Table 6, the negative chromatography step successfullyremoved a substantial amount of total protein while retaining asignificant amount of AAV. This increase of purity is also illustratedby the silver stain and Western blot in FIG. 6. The flow throughfraction comprised about 65% of the initial amount of AAV, whereas lessthan 6% of the AAV was present in the eluted fraction. Also, the totalprotein amount of the flow-through fraction was significantly reduced.Only about 34% of total protein remained in the FT fraction.

Description of Silver Stain

NuPAGE 4-12% Bis-Tris Midi Gel 1.0 mm. 20 well Cat. Nr. WG1402BX10MES SDS Running Buffer. Invitrogen. Cat. Nr. NP0002SB+DTT Incubation 10 min bei 70° C. 10 min cool down JAA treatment

Description of Western Blot

NuPAGE 4-12% Bis-Tris Midi Gel 1.0 mm. 20 well Cat. Nr. WG1402BX10MES SDS Running Buffer. Invitrogen. Cat. Nr. NP0002SB+DTT Incubation 10 min bei 70° C. 10 min cool down JAA treatment

1st Antibody: Mab to VP1. VP2 and VP3 of AAV (Adeno-Associated Virus)

Protein A affinity chromatography

PROGEN61058

2^(nd) Antibody: GOAT anti Mouse ALP

SIGMA A4656 1:2000 1 h Example 5

This example demonstrates an exemplary method of a second TFF step.

The AAV8-containing flow through (FT) fraction obtained through negativechromatography (Example 4) was concentrated and diafiltered against adiafiltration buffer (DB1) comprising 500 mM NaCl/50 mM TrisHCl; pH 8.5to condition the product for the following ultracentrifugation (UC) step(e.g., Example 6, 7, 9, 14, or 16). The second TFF step (TFF2) wascarried out using 4×0.1 m² Pall T-Series Omega Centramate Membranes 100kDa (4× Pall. Part No.: OS100T12) and the Pall UF/DF System CM500. Afterultra-centrifugation/diafiltration, the product was concentrated to atarget volume of 1200 ml and drained from the system. In order toincrease AAV8 yield the membrane and the UF/DF system were subsequentlyflushed with approx. 400 ml diafiltration buffer (10 min. recirculationon retentate side at 1 bar Feed pressure). The membrane flush and theconcentrated product were pooled and filtered using 0.2 μm Filter (PALLPart No.: KA02EAVP2S) for bioburden reduction. Table 7 details thescheme of TFF2 and Table 8 details the yield of this step.

TABLE 7 Pressure [bar] TMP (transmembrane Flux [l/h] Conductivity StepBuffer Volume Feed Retentate Filtrate pressure) Retentate Filtrate[mS/cm] Conditioning Diafiltration approx. 0.5-1.2 0.0-0.1 0.0 0.25-0.65n.a. n.a. n.a. and buffer 2000 ml Equilibration Ultrafiltration n.a. to1.6 0.1 0.0 0.85 188.4-190.8 61.0-77.5 14.84 (Concentration) 1600 mlDiafiltration Diafiltration 4-5 1.5-1.6 0.1 0.0 0.80-0.85 184.2-186.061.3-63.8 46.92 buffer Volume changes Ultrafiltration n.a. to 0.5-1.6n.a. n.a. n.a. n.a. n.a. n.a. (2^(nd) 1200 ml Concentration) Membraneflush Diafiltration ~400 ml 1.0 n.a. n.a. n.a. n.a. n.a. n.a. bufferDiafiltration buffer = 50 mM Tris/500 mM NaCl, pH 8.5 ± 0.2 bei 25° C.

TABLE 8 protein by protein Bradford protein by ITR OR- by Bradford ITRITR qPCR Volume* 1300448 Bradford Yield qPCR qPCR Yield Step [ml](μg/ml) (mg) (%) (vg/ml) (vg) (%) Load 22037.4 247 5437 100 4.61.E+111.02.E+16 100.00 Retentate 1600.0 1078 1725 32 5.88.E+12 9.41.E+15 92.61Filtrate 20301.5 171 3480 64 7.73.E+08 1.57.E+13 0.15 Diafiltrate 7716.5129 992 18 6.41.E+08 4.95.E+12 0.05 Retentate 1600.0 523 837 156.32.E+12 1.01.E+16 99.53 (diafiltrated) Retentate 1671.4 523 875 166.32.E+12 1.06.E+16 103.98 (diafiltrated) pooled with flush Prior 0.2 μmfiltration Retentate 1686.0 534 901 17 6.76.E+12 1.14.E+16 112.19(diafiltrated) pooled with flush Post 0.2 μm filtration HEK293 HEK293AAV AAV HEK293 HCP HCP AAV ELISA ELISA HCP ELISA ELISA ELISA total YieldELISA Total Yield Step (μg/ml) (mg) (%) (μg/ml) (mg) (%) Load 16.64366.81 100  122 2689  100 Retentate 194.27 310.84 85 356 570 21 Filtrate<0.03125 — — — — — Diafiltrate <0.03125 — — — — — Retentate 187.07299.31 82 101 162 6 (diafiltrated) Retentate 174.57 291.78 80 — — —(diafiltrated) pooled with flush Prior 0.2 μm filtration Retentate192.53 324.61 88 104 175 7 (diafiltrated) pooled with flush Post 0.2 μmfiltration

As shown in Table 8, the TFF2 step successfully reduced the volume toabout 7.5% of the original volume (load volume) while retaining 88% ofthe initial AAV.

Example 6

This example demonstrates an exemplary method of separating empty fromfull AAV particles through ultracentrifugation using 50% (w/w) bufferedethylene glycol solution and 50% (w/w) sucrose gradient. This stepadditionally removes host cell proteins (e.g., HSP70). Product relatedimpurities like “empty capsids” and host cell proteins such as HSP70 andLDH, also are eliminated via this step.

A 50% (w/w) ethylene glycol buffered solution in 50 mM TrisHCl/750 mMNaCl at pH 8.0 containing the AAV8 sample comprising vector DNA of humancoagulation Factor IX Padua in a single stranded self-complementary form(2.6 kb), was loaded into the ultracentrifuge (UC) followed by twodifferent sucrose solutions which vary in sucrose concentration. Thesucrose solution loaded into the UC first had a 55% sucroseconcentration. The sucrose solution loaded into the UC immediately afterthe first sucrose solution had a 60% sucrose concentration. The ratio ofloaded sample to sucrose gradient is 1:1 and sucrose solutions were ofequal volumes. The total core volume is 1,600 ml.

One kilogram of the buffered sucrose solution with a 55% sucroseconcentration was prepared by mixing 2.42 g ofTris(hydroxymethyl)aminomethane (Trometamol) with 8.00 g sodium chlorideand 550.00 g sucrose. WFI was added to near 1 kg, with 1 M NaOH and 25%HCl used to adjust the pH as needed. WFI was then added to 1 kg.

One kilogram of the buffered sucrose solution with a 60% sucroseconcentration was prepared by mixing 2.42 g ofTris(hydroxymethyl)aminomethane (Trometamol) with 8.00 g sodium chlorideand 600.00 g sucrose. WFI was added to near 1 kg, with 1 M NaOH and 25%HCl used to adjust the pH as needed. WFI was then added to 1 kg.

A density gradient forms during a first UC phase wherein rotationalspeed was set at 4,000 rpm and the temperature was maintained at 2-10°C. After the first phase, the rotational speed was increased to 35,000rpm and the temperature was increased to 22° C. During this second phaseat higher speed and temperature, full AAV particles were separated fromthe empty capsids. After 20 hours, the ultracentrifuge was stopped andthe fractions containing the majority of full AAV particles werecollected. Additional details relating to the UC step that wasperformed, including details about input parameters for building up thegradient, collecting the fraction containing the full particles, elutionrate, etc., are provided below in Table 9.

TABLE 9 Process Stage Parameter/Material Value General Equipment CC40Fermentation equivalent 1/2 of 500 L core volume 3.2 L Loading Loadingsequence AAV8 containing buffered ethylene glycol solution -> 55% Suc.-> 60% Suc. Volume of UFB-intermediate per one run 1.6 L Feed FlowrateUFB-intermediate 11.5 L/h Feed Flowrate Sucrose solutions 4.8 L/h Volumeof of 55% Sucrose Approx. 800 mL Volume of 60% Sucrose Approx. 800 mLConcentration of of Sucrose 55% Sucrose 55% ± 1% Concentration ofSucrose 60% Sucrose 60% ± 1% pH of of sucrose buffers (55%. 60%) 7.4 +/−0.3 Gradient build up Rotation speed: Increase from 0 rpm-4000 rpm Timefor gradient build up 17-25 min Chamber temperature during gradientbuild up 2-10° C. Acceleration Rotation speed: Increase from 4000-35000rpm Time for acceleration 5-60 min Chamber temperature duringacceleration 2-10° C. Separation Rotation speed 35000 ± 2000 rpm ChamberTemperature during separation Target: 22 +/− 2° C. Duration separation20 hours (can run 16-20 hours) Deceleration Rotation speed: Decreasefrom 35000-4000 rpm Time for deceleration 5-90 min Chamber temperatureduring deceleration 2-10° C. Fading out Rotation speed: Decrease from4000-0 rpm Time for fading out 15-30 min Chamber temperature duringfading out 2-10° C. Fractionation Elution flowrate 6 L/h Mass offraction 100-600 g Mass of peak pool (per Fermentation batch) 200-1800 gHolding time after UC - Temperature 2-8° C. Buffer pH & ConductivityComposition 55% sucrose solution pH 7.4 ± 0.2 at 25° C. 20 mM Tris. 8g/kg NaCl 55% sucrose 60% sucrose solution pH 7.4 ± 0.2 at 25° C. 20 mMTris. 8 g/kg NaCl 60% sucrose

The results are shown in FIG. 17, in which empty capsids (at fraction15) are resolved from the full capsids (at fraction 11).

FIG. 18 shows the results of undertaking four different assays on eachof the fractions shown in FIG. 17. qPCR refers to quantitative PCR ofITR of AAV8 (ITR-qPCR). AAV8:AG refers to ELISA against an AAV8 antigen.WAX % refers to the percentage of full capsids as quantified by weakanion exchange. DNA/AAV ratio (sometimes referred to as Spec. Act. orspecific activity herein), refers to the ratio of total vector genomes(ITR-qPCR) to the total amount of rAAV8 particles (AAV8:AG), which givesan indication as to the proportion of DNA-containing capsids as well asfull capsids. A higher DNA/AAV ratio is correlated with a higher amountof full capsids. The transition from full to empty capsids is seen bythe abrupt decline in Spec. Act. from fraction 13 to fraction 15. Thevalues for each assay were normalized to one another, as indicated bythe Y axis.

TABLE 10 Blue 06 DNA/AAV Fraction qPCR AAV8:AG WAX Ratio Nr.: vg/mlcp/ml [%] Full AAV8 vg/cp 1 2.61E+11  8.9E+11 86 2.93E+10 2 6.05E+111.99E+12 82 3.04E+10 3 1.08E+12 2.47E+12 82 4.37E+10 4 1.21E+12 3.32E+1285 3.64E+10 5 2.04E+12 4.94E+12 87 4.13E+10 6 2.51E+12 7.85E+12 903.20E+10 7   4E+12 1.57E+13 94 2.55E+10 8 6.41E+12 2.62E+13 96 2.45E+109  1.1E+13  4.4E+13 97 2.50E+10 10 1.86E+13 6.25E+13 98 2.98E+10 113.18E+13  8.4E+13 97 3.79E+10 12 4.19E+13  1.3E+14 97 3.22E+10 134.09E+13 1.395E+14  90 2.93E+10 14 2.69E+13 1.56E+14 76 1.72E+10 152.05E+13 1.79E+14 54 1.15E+10 16 1.18E+13 2.025E+14  42 5.83E+09 177.87E+12 1.985E+14  35 3.96E+09 18 6.28E+12  2.1E+14 39 2.99E+09 195.06E+12 1.43E+14 36 3.54E+09 20  3.1E+12 8.79E+13 38 3.53E+09

Example 7

This example demonstrates an exemplary method of separating empty fromfull AAV particles through ultracentrifugation using a sucrose gradient.This step additionally removed host cell proteins (e.g., HSP70). Productrelated impurities like “empty capsids” (as defined above) also wereeliminated via this step.

The concentrated fraction containing vector DNA comprising singlestranded DNA encoding B-Domain deleted human coagulation factor VIIIobtained via TFF2 of Example 5 was loaded into the ultracentrifuge (UC)followed by three different sucrose solutions which vary in sucroseconcentration. The sucrose solution loaded into the UC first had a 50%sucrose concentration. The sucrose solution loaded into the UCimmediately after the first sucrose solution had a 55% sucroseconcentration and the sucrose solution loaded into the UC last had a 60%sucrose concentration.

One kilogram of the buffered sucrose solution with a 50% sucroseconcentration was prepared by mixing 2.42 g ofTris(hydroxymethyl)aminomethane (Trometamol) with 8.00 g sodium chlorideand 500.00 g sucrose. WFI was added to near 1 kg, with 1 M NaOH and 25%HCl used to adjust the pH as needed. WFI was then added to 1 kg.

One kilogram of the buffered sucrose solution with a 55% sucroseconcentration was prepared by mixing 2.42 g ofTris(hydroxymethyl)aminomethane (Trometamol) with 8.00 g sodium chlorideand 550.00 g sucrose. WFI was added to near 1 kg, with 1 M NaOH and 25%HCl used to adjust the pH as needed. WFI was then added to 1 kg.

One kilogram of the buffered sucrose solution with a 60% sucroseconcentration was prepared by mixing 2.42 g ofTris(hydroxymethyl)aminomethane (Trometamol) with 8.00 g sodium chlorideand 600.00 g sucrose. WFI was added to near 1 kg, with 1 M NaOH and 25%HCl used to adjust the pH as needed. WFI was then added to 1 kg.

A density gradient formed during a first UC phase wherein rotationalspeed is set at 4000 rpm and the temperature was maintained at 2-10° C.After the first phase, the rotational speed was increased to 35000 rpmand the temperature increased to 22° C. During this second phase athigher speed and temperature, full AAV particles were separated from theempty capsids. After approximately 5 hours, the ultracentrifuge wasstopped and the fractions containing the majority of full AAV particleswere collected. Additional details relating to the UC step that wasperformed, including details about input parameters for building up thegradient, collecting the fraction containing the full particles, elutionrate, etc., are provided below in Tables 11 and 12.

TABLE 11 Process Stage Parameter/Material Value General Equipment CC40SFermentation equivalent 200 L core volume 3.2 L Loading Loading sequenceUFB-intermediate -> 50% Suc. -> 55% Suc. -> 60% Suc. Volume ofUFB-intermediate per one run 1.6 L Feed Flowrate UFB-intermediate 6 L/hFeed Flowrate Sucrose solutions 1.5 L/h Volume of 50% Sucrose 400 ± 20mL Volume of 55% Sucrose 200 ± 10 mL Volume of 60% Sucrose 200 ± 10 mLConcentration of Sucrose 50% Sucrose 50% ± 1% Concentration of Sucrose55% Sucrose 55% ± 1% Concentration of Sucrose 60% Sucrose 60% ± 1% pH ofsucrose buffers (50%. 55%. 60%) 7.4 +/− 0.3 Gradient build up Rotationspeed: Increase from 0 rpm-4000 rpm Time for gradient build up 17-25 minChamber temperature during gradient build up 2-10° C. AccelerationRotation speed: Increase from 4.000-35.000 rpm Time for acceleration5-60 min Chamber temperature during acceleration 2-10° C. SeparationRotation speed 35.000 ± 2.000 rpm Chamber Temperature during separationTarget: 22 +/− 2° C. Duration separation 300 ± 30 min DecelerationRotation speed: Decrease from 35.000-4.000 rpm Time for deceleration5-90 min Chamber temperature during deceleration 2-10° C. Fading outRotation speed: Decrease from 4.000-0 rpm Time for fading out 15-30 minChamber temperature during fading out 2-10° C. Fractionation Elutionflowrate 3 L/h Volume of fraction 25-50 mL Volume of peak pool (perFermentation batch) 300-450 mL Fraction start density Brix 56.5-54Fraction start (UV 254) increase >0.004 Fraction end (UV 254) after peakmaximum of UV254 and no decrease of signal Fraction end density Brix 51Holding time after UC - Temperature 2-8° C. Holding time after UC - Timemax.7 days Buffer pH & Conductivity Composition 50% sucrose solution pH7.4 ± 0.2 at 25° C. 20 mM Tris. 8 g/kg NaCl 50% sucrose 55% sucrosesolution pH 7.4 ± 0.2 at 25° C. 20 mM Tris. 8 g/kg NaCl 55% sucrose 60%sucrose solution pH 7.4 ± 0.2 at 25° C. 20 mM Tris. 8 g/kg NaCl 60%sucrose

TABLE 12 ITR qPCR AAV ELISA HEK293 HCP ELISA Volume Total Yield TotalYield Total Yield Step [ml] (vg/ml) (vg) (%) (μg/ml) (mg) (%) (μg/ml)(mg) (%) Load 1600 7.8E+12 1.3E+16 100 206.4 330 100 95.9 153.44 100Forerun 300 — — — 4.1 1 0 — — — Peak Pool 250 1.6E+13 3.9E+15  31 83.721 6 <0.5 0.125 0.08147 Mostly empty 350 — — — 719.6 252 76 — — —capsids Tail run 2300 — — — 10.7 25 7 — — — HEK293 DNA PCR In vitro WAXVolume Total Yield Biopotency (Area % peak Step [ml] (ng/ml) (μg) (%)(BPU) 2) Load 1600 5 8 100 0.65 n.d. Forerun 300 — — — — Peak Pool 2501.35 0.3375 0.10 1.25 82% Mostly empty 350 — — — — — capsids Tail run2300 — — — — —

The elution pattern is presented in FIG. 8 and silver stain and westernblots of the load and single fractions are presented in FIG. 9. As shownin Table 12, 82% of the particles in the peak pool are considered asfull particles.

Description of Silver Stain

NuPAGE 4-12% Bis-Tris Midi Gel 1.0 mm. 20 well Cat. Nr. WG1402BX10MES SDS Running Buffer. Invitrogen. Cat. Nr. NP0002SB+DTT Incubation 10 min at 70° C. 10 min cool down JAA treatment

Description of Western Blot

NuPAGE 4-12% Bis-Tris Midi Gel 1.0 mm. 20 well Cat. Nr. WG1402BX10MES SDS Running Buffer. Invitrogen. Cat. Nr. NP0002SB+DTT Incubation 10 min at 70° C. 10 min cool down JAA treatment

1st Antibody: Mab to VP1. VP2 and VP3 of AAV (Adeno-Associated Virus)

Protein A affinity chromatography

PROGEN61058

2^(nd) Antibody: GOAT anti Mouse ALP

SIGMA A4656 1:2000 1 h Example 8

This example demonstrates an exemplary method of inactivating lipidenveloped viruses and a polish step.

The AAV8-containing fractions obtained upon the UC step of Example 7were pooled and diluted 1:2.5 with an equilibration buffer (50 mM Tris;pH 8.5±0.2). Treatment with a solvent detergent (0.3%Tri-n-butylphosphate; 1.0% Triton X100; 0.3% Polysorbate 80) wasperformed. The conductivity of the solvent was subsequently adjusted to3.6±0.2 mS/cm with a further 1:2 dilution. The resulting solution wasloaded onto a chromatography column containing Fractogel® EMD TMAE (M)(Merck-Millipore Cat.: 116881) ID 22 mm bed height 57 mm(Merck-Millipore Vantage VL 22x250. Cat. Nr.: 96220250), and then washedwith equilibration buffer (50 mM Tris pH 8.5±0.2). A second wash stepwas performed with a buffer (8.09 mM Na₂HPO₄; 1.47 mM KH₂PO₄; 2.68 mMKCl; 5% Sorbitol). The column elution was conducted by a NaCl gradientof 12 column volumes—from a first elution buffer comprising 8.09 mMNa₂HPO₄; 1.47 mM KH₂PO₄; 2.68 mM KCl; 5% Sorbitol to a last elutionbuffer comprising 8.09 mM Na₂HPO₄ 1.47 mM KH₂PO₄; 2.68 mM KCl; 5%Sorbitol+600 mM NaCl. The main amount of the AAV8 elutes as a distinctsharp peak and was collected as E2 (Eluate2) which represents thepurified AAV8 product. The column was finally stripped with 2M sodiumchloride followed by a regeneration procedure. Tables 13 and 14 detailthese steps and the yield obtained after performing these steps. FIG. 10is the final polishing of AAV8 on Fractogel TMAE.

TABLE 13 Step Buffer Inlet Flowrate CV Outlet Fraction Gel activationTWA-buffer A13 60 cm/h  5 F1 Waste Equilibration T50EQ-buffer A11 10 F1Waste Sample load Sample-Load A2 — F3 ABS With air sensorReEquilibration T50EQ-buffer A11 10 F3 ABS Wash Fractogel-equilibrationbuffer A12 10 F4 WASH Elution: Fractogel-Equilibration buffer A12 12 F2diverse Gradient from A12 to B1 Elution buffer B1 (Frac in 12CV Elution:Elution buffer B1 (100%)  3 F2 diverse (Frac) BUFFER T50EQ-bufferEquilibration buffer 50 mM Tris pH 8.5 ± 0.2 Fractogel-Equilibrationbuffer 20 mM Sodium phosphate/2.68 mM Potassium chloride/pH 8.0 ± 0.2 +5% Sorbitol Elution buffer 15 mM Sodium phosphate/5 mM Potassiumphosphate/2.68 mM Potassium chloride/ 600 mM NaCl/pH 7.4 ± 0.2 + 5%Sorbitol

TABLE 14 YIELD Volume qPCR FVIII AAV8 ELISA DNA/AAV (g) vg/mL vg % μg/mLμg % ratio vg/μg L Dil 611.91 3.25E+12 1.99E+15 100.00 17.542 10734.04100.00 1.85E+11 E 32.86 4.59E+13 1.51E+15 75.84 273.857 8998.94 83.841.68E+11 Bulk 59.34 1.89E+13 1.12E+15 74.36 122.89 7292.29 81.041.54E+11 Volume HEK293 HCP ELISA HEK-DNA (g) μg/mL μg % ng/mL DNA/AAVratio vg/μg L Dil 611.91 <0.2 <122.38 — — <0.01 E 32.86 — — — — — Bulk59.34 <0.1  <5.93 — 2.8 <0.01 L Dil: is the diluted pool fromultracentrifugation prior the SD treatment and the final dilution rate;E is the Eluate from the AEX column; Bulk is the 1:2 diluted Eluate fromthe AEX column. Bulk is the formulated product prior dilution to thefinal drug product (FDP)

Example 9

This example demonstrates an exemplary method of purifying AAV fromunpurified fractions comprising an ultracentrifugation step during whicha density gradient forms.

FIG. 11 is a schematic of the steps carried out in the exemplary method.The ultracentrifugation step was performed using a core with a capacityfor 3.2 L volume (1.6 L product+1.6 L gradient/TBS buffer).

With the UC in standstill, the 1.6 L starting material (intermediateproduct from the previous process step=UFB) was loaded into the rotorwith a constant flow rate of 6 L/h followed by the load of 3 sucrosesolutions of varying density. The sucrose solutions were loaded with aconstant flow rate of 1.5 L/h in the following order: 800 mL of 50%sucrose, 400 mL of 55% sucrose and 400 mL of 60% sucrose.

The filled rotor was accelerated from 0 up to 4000 rpm in order toachieve the zonal mode to build up a sucrose gradient with increasingdensity, followed by a second acceleration from 4000 up to 35000 rpm.The UC runs at 35000 rpm (approx. 90000 g-force) for 5 hours. Once thefinal speed was achieved, the temperature was shifted from +4° C. to+22° C. The AAV8 particles contained in the starting material moves intothe sucrose gradient until they reach a point at which their densitymatches with the density of the surrounding gradient. As full and emptycapsids differ in their density, this feature is used for the separationof full from empty virus capsids. One hour before the centrifugationstep was finished, the temperature was manually shifted from +22° C. to+4° C. Stopping the UC from 35,000 to 0 rpm was performed in 2 steps:from 35,000 to 4,000 rpm by deceleration and from 4,000 rpm to 0 rpm byletting the rotor fade out.

The product was recovered with a flow rate of 6 L/h by collecting 25 mLfractions as well as a waste fraction at the end of the elution.

The collection of fractions 25 mL each starts with fraction 7 and endsat the beginning of the waste peak. The first peak in FIG. 12 at ahigher sucrose density represents the “Full capsids”. The second peakrepresents the “mostly empty capsids”.

Fractions F10 to F14 were pooled and further purified on Fractogel® EMDTMAE (M) as standard AAV8 “Full capsids” in the same way as the otherAAV8 containing fractions. Table 15 provides details of the TMAEchromatography step and Table 16 details the buffers used.

TABLE 15 Step Buffer Amount Flow rate 1 Equilibration 1 TWA (2M NaCl) 5100 2 Equilibration 2 T50EQ 10 100 3 Product load diluted SD treated100-300 mL 100 UC pool Cond: 2.5-4.5 mS/cm pH 8.3-8.7 No filtration 4Wash 1 20 100 5 Wash 2 10 60 6 Elution Gradient 0-100% 12 60 Chat_EL inChat_A Pool collecting Start: UV280 > 0.02 AU, End at 17% of peak max(UV280) decreasing 7 Regeneration x

TABLE 16 Buffer Buffer content pH Conductivity Equilibration 1 2 molNaCl n.d 140-172 mS/cm/ 25° C. Equilibration 2 50 mM TrisHCl 8.5 ± 0.21-2 mS/cm/25° C. and Wash 1 Wash 2 8.09 mM Na2HPO4, 7.4 ± 0.2 1-2mS/cm/25° C. 1.47 mM KH2PO4, 2.68 mM KCl, 5% Sorbitol Elution 8.09 mMNa2HPO4, 7.4 ± 0.2 51-55 mS/cm/ 1.47 mM KH2PO4, 25° C. 2.68 mM KCl, 5%sorbitol + 600 mM NaCl

Approximately 25 ml of a single ultracentrifugation fraction was appliedseparately to a 4 ml TMAE Column by dilution of 1:2.5 with theequilibration buffer 50 mM Tris, pH 8.5±0.2. After the solvent detergenttreatment in the presence of 0.3% Tri-n-butylphosphate, 1% Triton X-100and 0.3% Polysorbate 80 the conductivity was adjusted to 3.6±0.2 mS/cmwith a further 1:2 dilution and the AAV8 containing solution was appliedto a chromatography column containing Fractogel® EMD TMAE (M)(Merck-Millipore, cat. no. 116881) ID 10 mm, bed height 50 mm±5 mm(Merck-Millipore Vantage VL 10x250 or similar) followed by a first washstep with equilibration buffer 50 mM Tris, pH 8.5±0.2 and a second washstep with 8.09 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 2.68 mM KCl, 5% sorbitol, pH7.4. The elution is conducted by a gradient of 12 column volumes from8.09 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 2.68 mM KCl, 5% sorbitol, pH 7.4 to8.09 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 2.68 mM KCl, 5% sorbitol+600 mM NaCl,pH 7.4. The main amount of the AAV8 elutes as a distinct sharp peak andwas collected as E2 (Eluate2) which represents the purified AAV8product. The obtained AAV8 containing eluate E2 (Eluate2) was diluted1:2 with 8.09 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 2.68 mM KCl, 5% sorbitol+600mM NaCl pH 7.4 to adjust to the matrix of the formulation buffer (8.09mM Na₂HPO₄, 1.47 mM KH₂PO₄, 2.68 mM KCl, 5% sorbitol+350 mM NaCl, pH7.4). The column was finally stripped with 2 M sodium chloride followedby a regeneration procedure. The chromatography system was placed at anambient temperature of +18° C. to +25° C.

Fractions 10 to 21 were analyzed with analytical ultracentrifugation(AUC) and DNA-Analysis of AAV8-vectors (Containing Factor IX Paduadouble stranded (4.8 kb)) was performed by native agarose gelelectrophoresis and alkaline agarose gel electrophoresis. FIGS. 13-16show analytical data of single fractions obtained by UC after polishingon TMAE. FIG. 14 is a photo of an Agarose 1% native and FIG. 15 is aphoto of an Agarose 0.8% alkaline. As shown in FIG. 15, the pooledfractions X10 to X14 of the UV peak obtained at higher sucrose densityin the ultracentrifugation contained a highly pure single DNA bandwithout any further hint to smaller DNA variants of the vector DNA.

Data of analytic UC is provided in Table 17.

TABLE 17 partially Fraction Number empty (50S) filled filled (80S)F10-14 4% 0% 96% F15 6% 1% 93% F16 16% 0% 85% F17 21% 0% 79% F18 29% 23%48% F19 41% 35% 24% F20 52% 43% 5% F21 60% 38% 3% F25 82% 17% 1% F26 81%18% 2% Fraction Nos. 22, 23 and 24 not analyzed

As shown in Table 17, Fractions 10-15 contained the highest amounts offilled AAV capsids. While substantial amounts of full capsids are foundin Fractions 16 and 17, the amounts were slightly less and the amount ofempty capsids rises, relative to Fractions 10-15. A further decrease inthe amount of full capsids and further increase in empty capsids wereobserved in Fractions 19 and 20.

FIG. 16 shows data for single BDS from indicated UC fractions. Theseresults show a homogenous AAV8 product from UC fraction 10 to 15 with nodetection of incomplete vector DNA content in AUC and agarose gelelectrophoresis. Incomplete vector DNA appears from fraction 17 and wasdetected in later fractions of the “mostly empty capsids” zone. Thedetection of incomplete vectors was surprising because one would notexpect to have such a wide range of resolution. Thus, the developedseparation method is not limited to resolving capsids that arecompletely empty (i.e. no DNA) from those comprising “full”, but itsurprisingly also allows the separation of vector DNA of varying lengthsthat cannot be resolved from one another. It is also an indication thatthere is a meaningful separation of “full capsids” from “empty capsids”regardless of the length of the DNA in the “empty capsid” population. Inother words, this method allows a depletion of empty vectors (includingtruncated and incomplete DNA) to obtain AAV with a high content of fullAAV capsids.

Example 10

This method describes an exemplary method of nanofiltration for removalof viruses larger than AAV and larger than the pore size of thenanofilter applied.

An Asahi PLANOVA 35 nm filter, which contains bundles of micro-poroushollow-fibers constructed of natural hydrophilic cuprammoniumregenerated cellulose with a narrow pore size range of 35 nm+/−2 nm, istested with an integrity leakage test to confirm that the filter is freefrom pinholes or large defects and pre-washed with formulation buffer.Then anion exchange eluate is taken and filtered under constant pressurenot exceeding 0.1 MPa in a dead-end mode through the nanofilter.Alternatively the filter could be run also in tangential flow method.Anion exchange eluate may be pre-diluted with elution buffer (PBS+600 mMNaCl) to adjust concentration of the virus particles. In order torecover all virus particles the nanofilter is post-washed with buffer(e.g. formulation buffer). Post-integrity testing of the nanofilter isperformed with the leakage test and a gold particle test to prove thatthe nanofilter pore size distribution has not changed and the nanofilterremained its integrity during filtration. Higher sample load results inhigher yield. Therefore, typically more than 50 L of solution per m²filter area is applied.

Example 11

This method describes an exemplary method of nanofiltration for removalof viruses larger than AAV and larger than the pore size of thenanofilter applied.

An Asahi PLANOVA 35 nm filter, which contains bundles of micro-poroushollow-fibers constructed of natural hydrophilic cuprammoniumregenerated cellulose with a narrow pore size range of 35 nm+/−2 nm, wastested with an integrity leakage test to confirm that the filter is freefrom pinholes or large defects and pre-washed with formulation buffer.Then, 4.05 ml of the pooled fractions comprising 2870.98 μg/ml AAV-8(concentration determined by ELISA) was first diluted in 4.05 of abuffer of 1.47 mM KH₂PO₄, 2.68 mM KCl, 8.09 mM Na₂HPO₄, 600 mM NaCl and5% Sorbitol, at pH 7.4. The dilution factor was 1:2. Then, 8.10 grams ofthe first dilution was diluted in 61.57 grams of a buffer of 1.47 mMKH₂PO₄, 2.68 mM KCl, 8.09 mM Na₂HPO₄, 350 mM NaCl and 5% Sorbitol, at pH7.4 to yield a load buffer. The dilution factor of the load buffer was1:8.6 as compared to the first dilution, and 1:17.2 as compared to thepooled fractions.

The nanofilter was conditioned with 25 ml of a buffer comprising 1.47 mMKH₂PO₄, 2.68 mM KCl, 8.09 mM Na₂HPO₄, 350 mM NaCl and 5% Sorbitol, at pH7.4. During the conditioning, 10 ml of the buffer was used to purge thefilter without pressure applied and with the retentate outlet open.Then, about 15 ml of the buffer was filtrated through the hollow fibers,with 0.9 bar of constant pressure applied and the retentate outletclosed.

Then, 9 ml of a flushing buffer was applied to the reservoir tank under0.9 bar of constant pressure. The flushing buffer comprised 1.47 mMKH₂PO₄, 2.68 mM KCl, 8.09 mM Na₂HPO₄, 350 mM NaCl and 5% Sorbitol, at pH7.4. Samples were then taken after flushing. After samples were taken,60 grams of the above load buffer was then filtered under constantpressure of 0.9 bar in a dead-end mode through the nanofilter.

Data from the filtration is presented in Table 18 below. The yield ofAAV from filtration is at least 77%.

TABLE 18 AAV AAV AAV Load ELISA ELISA ELISA AAV Volume GatternigGatternig AAV ELISA AAV ELISA Weber [mg] [g] or Total Yield Weber WeberYield bzw. Sample code: [ml] (μg) (%) (particles/ml) (particles) (%)[g/m²] AV_meC_NFA_04A_POOL 4.05 10955.747 — 9.19E+14 3.72E+15 — —AV_meC_NFA_04A_LOAD 60.00 10111.686 100.00 5.35E+13 3.21E+15 100.0010.112 AV_meC_NFA_04A_NF 60.25 7659.631 75.75 4.13E+13 2.49E+15 77.52 —AV_meC_NFA_04A_FLUSH 9.34 601.920 5.95 2.09E+11 1.95E+12 0.06 —AV_meC_NFA_04A_BULK 66.63 8392.144 82.995 3.73E+13 2.49E+15 77.424 —

Example 12

The following example describes an exemplary method of testingbiopotency of an AAV8 product.

In the in-vitro biopotency assay, the viral vector AAV8-FIX transfect ahepatic target cell line, which subsequently secretes functional,measurable FIX protein into the medium.

In a first step HepG2 target cells are transduced by AAV8-FIX. Duringincubation time FIX is released into cell supernatant. In a second step,the factor IX-activity of the cell culture supernatant is directlymeasured by a FIX chromogenic assay. The measurement of an AAV8-FIXsample is given as percentage relative to a reference material. Themethod allows a quantitative assessment of the biologic function of theAAV8-FIX gene therapy vector.

Example 13

This example demonstrates an AAV-specific ELISA.

In the foregoing examples, an AAV-specific ELISA was carried out aftersteps of the process of the present disclosure to identifyAAV-containing fractions and to calculate the DNA/AAV ratio or “Specificactivity” of the AAV which is represented by the ratio of qPCR to μgAAV8. The DNA/AAV ratio reflects the vector DNA encapsulated in the AAVparticles.

AAV8 ELISA was carried out with an AAV-8 titration ELISA Kit (Art. No.PRAAV8; Progen (Heidelberg, Germany) on a TECAN Roboter system. Briefly,a monoclonal antibody specific for a conformational epitope on assembledAAV8 capsids (ADK8) was coated onto microtiter strips and was used tocapture AAV8 particles from the AAV fraction. The capture AAV8 particleswere detected by two steps. In a first step, a biotin-conjugatedmonoclonal antibody specific for the ADK8 antibody was bound to theimmune complex (of ADK8 and ADK8 antibody). Streptavidin peroxidaseconjugates were added to the immune complexes bound to thebiotin-conjugated monoclonal antibody and the streptavidin peroxidaseconjugates reacted with the biotin. A peroxidase substrate solution wasadded and a color reaction which is proportional to the amount of boundAAV particles occurs. The color reaction is measured photometrically at450 nm.

The ELISA determines the amount of AAV8 particles present in the testedfraction.

Example 14

This example demonstrates an exemplary method of separating empty fromfull AAV particles through ultracentrifugation using a 50% ethyleneglycol buffered loading solution and 50% sucrose gradient (i.e., 1:1ratio). Product related impurities like “empty capsids” and host cellproteins such as HSP70 and LDH, are eliminated via this step.

The buffered ethylene glycol solution (50% (w/w) in TrisHCl/NaCl)comprising AAV8, with human coagulation Factor VIII (full length ˜4.8kb), was loaded into the ultracentrifuge (UC) followed by a sucrosegradient formed of two different sucrose solutions which vary in sucroseconcentration. The ratio of loaded sample to sucrose gradient is 1:1.The sucrose solution loaded into the UC first had a 55% sucroseconcentration. The sucrose solution loaded into the UC immediately afterthe first sucrose solution had a 60% sucrose concentration.

One kilogram of the buffered sucrose solution with a 55% sucroseconcentration was prepared by mixing 2.42 g ofTris(hydroxymethyl)aminomethane (Trometamol) with 8.00 g sodium chlorideand 550.00 g sucrose. WFI was added to near 1 kg, with 1 M NaOH and 25%HCl used to adjust the pH as needed. WFI was then added to 1 kg.

One kilogram of the buffered sucrose solution with a 60% sucroseconcentration was prepared by mixing 2.42 g ofTris(hydroxymethyl)aminomethane (Trometamol) with 8.00 g sodium chlorideand 600.00 g sucrose. WFI was added to near 1 kg, with 1 M NaOH and 25%HCl used to adjust the pH as needed. WFI was then added to 1 kg.

In this example, a 3,200 ml core was used, wherein the 50% ethyleneglycol buffered solution containing AAV8 was 1,600 ml, the 55% (w/w)sucrose solution was 800 ml, and the 60% (w/w) sucrose solution was 800ml.

A density gradient forms during a first UC phase wherein rotationalspeed was set at 4000 rpm and the temperature was maintained at 2-10° C.After the first phase, the rotational speed was increased to 35000 rpmand the temperature was increased to 22° C. During this second phase athigher speed and temperature, full AAV particles were separated from theempty capsids. After 20 hours, the ultracentrifuge was stopped and thefractions containing the majority of full AAV particles were collected.

The results are shown in FIG. 19, in which full capsids (fractions 1-6)are resolved from the empty capsids (fractions 8-17).

FIG. 20 is an overlay of sedimentation coefficient graphs from Fractions8-10 demonstrating subspecies (i.e., incomplete vector DNA) separation.The arrow denotes a shift towards AAV8 with lower weight from fractionswith higher density to lower density in the UC-gradient (i.e., density:Fraction 8>Fraction 9>Fraction 10). This is further demonstration thatvector DNA can be separated based on size with appropriate resolutionwith this protocol. See also, Table 19.

TABLE 19 AUC Area AUC Area WAX WAX [%] [%] AUC Area [%] [%] emptyintermediate1 [%] Full full empty (50S) (60S) (70-80S) Pool Fraction 955 5.6 4.3 79.2 01 to 06 Fraction 08 80 20 1.5 7.4 87.5 Fraction 09 65 3516.7 2.2 81.1 Fraction 10 40 60 57.6 1.8 40.6

Example 15

This example demonstrates ultracentrifugation using a sucrose gradient,using the 50-55-60 sucrose solution method. The method is the same asindicated in Example 7, except the AAV8 particles contained singlestranded vector DNA of human coagulation Factor IX Padua (2.6 kB). Asindicated in FIG. 21, the separation between full versus empty capsidsis not as resolved as indicated by the overlap of the ITR pPCR and AAVELISA.

Example 16

This example demonstrates ultracentrifugation using a sucrose gradient,using the 55-60 sucrose solution method as described in Example 6,except using a total of 7,700 ml core volume. Thus, the load volume was6,100 ml in 50% ethylene glycol TrisHCl buffered solution, the 55%sucrose solution volume was 800 ml, and the 60% sucrose solution wasalso 800 ml. The ultracentrifugation elution profile is depicted in FIG.22. Here there are no “waste peaks” because the AAV is highly purified.Full AAV8 is collected from Fractions 1-8 (50 ml each). The “empty” AAVis collected from Fractions 9-15 (50 ml each). Further fractions, 15-21are also collected (100 ml each). The first peak in FIG. 22 at a highersucrose density represents the “Full capsids”. The second peakrepresents the “empty capsids”.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the disclosure (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range and each endpoint, unless otherwise indicatedherein, and each separate value and endpoint is incorporated into thespecification as if it were individually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate thedisclosure and does not pose a limitation on the scope of the disclosureunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the disclosure.

Preferred embodiments of this disclosure are described herein, includingthe best mode known to the inventors for carrying out the disclosure.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the disclosure to be practicedotherwise than as specifically described herein. Accordingly, thisdisclosure includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the disclosure unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed is:
 1. A method of purifying full adeno-associated virus(AAV) capsids from a concentrated AAV fraction comprising empty AAVcapsids and full AAV capsids, comprising: (i) loading into a zonal rotora) the concentrated AAV fraction and b) at least two sugar solutions,each of which has a different sugar concentration and each of whichcomprises a sugar at a concentration equivalent to a sucroseconcentration ranging from about 45% (w/w) to about 65% (w/w) sucrose,(ii) operating an ultracentrifuge comprising the zonal rotor in batchmode, whereupon a sugar gradient is formed, (iii) obtaining a fractionof the sugar gradient to obtain an AAV fraction wherein at least orabout 60% of the AAV particles in the AAV fraction are full AAV capsids,wherein (A) the volume of the sugar solutions is greater than or equalto about 50% of the volume of the zonal rotor, (B) the total volume ofthe sugar solutions and the AAV fraction is less than or equal to thevolume of the zonal rotor, (C) the ratio of the volume of the sugarsolutions to the volume of the AAV fraction is less than or equal toone, or (D) a combination thereof.
 2. The method of claim 1, whereineach sugar solution comprises a sugar at a concentration equivalent to asucrose concentration ranging from about 50% (w/w) to about 60% (w/w)sucrose.
 3. The method of claim 1 or claim 2, wherein each sugarsolution comprises a sugar at a concentration equivalent to a sucroseconcentration ranging from about 55% (w/w) to about 60% (w/w) sucrose.4. The method of claim 1 or claim 2, wherein at least one of the sugarsolutions comprises sugar at a concentration equivalent to a sucroseconcentration greater than about 50% (w/w) sucrose.
 5. The method of anyone of claims 1-4, wherein at least one of the sugar solutions comprisessugar at a concentration equivalent to a sucrose concentration greaterthan about 55% (w/w) sucrose.
 6. The method of any one of claims 1-5,wherein at least one of the sugar solutions comprises sugar at aconcentration equivalent to a sucrose concentration ranging from about60% (w/w) to about 65% (w/w) sucrose.
 7. The method of any one of claim1, 2, or 4-6, wherein one sugar solution comprises a sugar at aconcentration equivalent to a sucrose concentration ranging from about52% (w/w) to about 58% (w/w) sucrose, wherein a second sugar solutioncomprises a sugar at a concentration equivalent to a sucroseconcentration ranging from about 57% (w/w) to about 63% (w/w) sucrose,and optionally wherein a third sugar solution comprises a sugar at aconcentration equivalent to a sucrose concentration ranging from about47% (w/w) to about 53% (w/w) sucrose.
 8. The method of any one of claims1-7, wherein two sugar solutions are loaded into the zonal rotor.
 9. Themethod of any one of claim 1, 2, or 4-8, wherein two sugar solutions areloaded into the zonal rotor and wherein one sugar solution comprises asugar at a concentration equivalent to a sucrose concentration rangingfrom about 52% (w/w) to about 58% (w/w) sucrose and a second sugarsolution comprises a sugar at a concentration equivalent to a sucroseconcentration ranging from about 57% (w/w) to about 63% (w/w) sucrose.10. The method of any one of claims 1-7, wherein at least three sugarsolutions are loaded into the zonal rotor.
 11. The method of any one ofclaim 1-7 or 10, wherein three sugar solutions are loaded into the zonalrotor.
 12. The method of any one of claim 1, 2, 4-7, 10, or 11, whereinthree sugar solutions are loaded into the zonal rotor and wherein onesugar solution comprises a sugar at a concentration equivalent to asucrose concentration ranging from about 47% (w/w) to about 53% (w/w)sucrose, a second sugar solution comprises a sugar at a concentrationequivalent to a sucrose concentration ranging from about 52% (w/w) toabout 58% (w/w) sucrose, and a third sugar solution comprises a sugar ata concentration equivalent to a sucrose concentration ranging from about57% (w/w) to about 63% (w/w) sucrose.
 13. The method of any one of claim1, 2, or 4-12, wherein in step (i), the concentrated AAV fraction isloaded before a sugar solution comprising a sugar at a concentrationequivalent to a sucrose concentration ranging from about 52% (w/w) toabout 58% (w/w) sucrose, and wherein the sugar solution comprising asugar at a concentration equivalent to a sucrose concentration rangingfrom about 52% (w/w) to about 58% (w/w) sucrose is loaded before a sugarsolution comprising a sugar at a concentration equivalent to a sucroseconcentration ranging from about 57% (w/w) to about 63% (w/w) sucrose.14. The method of any one of claim 1, 2, 4-7, or 10-13, wherein in step(i), the concentrated AAV fraction is loaded before a sugar solutioncomprising a sugar at a concentration equivalent to a sucroseconcentration ranging from about 47% (w/w) to about 53% (w/w) sucrose,wherein the sugar solution comprising a sugar at a concentrationequivalent to a sucrose concentration ranging from about 47% (w/w) toabout 53% (w/w) sucrose is loaded before a sugar solution comprising asugar at a concentration equivalent to a sucrose concentration rangingfrom about 52% (w/w) to about 58% (w/w) sucrose, and wherein the sugarsolution comprising a sugar at a concentration equivalent to a sucroseconcentration ranging from about 52% (w/w) to about 58% (w/w) sucrose isloaded before a sugar solution comprising a sugar at a concentrationequivalent to a sucrose concentration ranging from about 57% (w/w) toabout 63% (w/w) sucrose.
 15. The method of any one of claims 1-14,wherein each sugar solution is loaded in the zonal rotor at equalvolumes.
 16. The method of any one of claims 1-14, wherein the sugarsolution with the smallest sugar concentration is loaded in the zonalrotor at a volume which is twice the volume of at least one of the othersugar solutions in the zonal rotor.
 17. The method of any one of claim1-14 or 16, wherein the sugar solution with the smallest sugarconcentration is loaded in the zonal rotor at a volume which is at leastthe volume of all other sugar solutions combined in the zonal rotor. 18.The method of claim 1-14, 16, or 17, wherein the sugar solution with thesmallest sugar concentration is loaded in the zonal rotor at a volumewhich is at least twice the volume of the sugar solution with thelargest sugar concentration, optionally, wherein the volume of the sugarsolution with the largest sugar concentration is equal to the volume ofthe sugar solution with the intermediate sugar concentration.
 19. Themethod of any one of claim 1-14 or 16-18, wherein at least two sugarsolutions are loaded into the zonal rotor, wherein the sugar solutionwith the smallest sugar concentration is loaded in the zonal rotor at avolume which is at least twice the volume of at least one other sugarsolution in the zonal rotor.
 20. The method of any one of claims 1-19,wherein at least two sugar solutions are loaded into the zonal rotor,wherein the sugar solution with the smallest sugar concentration isloaded in the zonal rotor at a volume which is the same volume of atleast one other sugar solution in the zonal rotor.
 21. The method of anyone of claim 1-20 wherein the sugar solution with the smallest sugarconcentration is loaded in the zonal rotor at a volume which is half thevolume of the concentrated AAV fraction.
 22. The method of any one ofclaims 1-21, where in the ratio of the volume of the total sugargradient to the volume of the AAV fraction loaded in the zonal rotor isfrom about 1:1 to about 1:5.
 23. The method of any one of claims 1-22,wherein the AAV fraction comprises a buffered solution.
 24. The methodof claim 23, wherein the buffered solution comprises TrisHCl and NaCl.25. The method of claim 24, wherein the TrisHCl is at a concentration ofabout 20 to about 50 mM and the NaCl is at a concentration of about 150mM to about 900 mM.
 26. The method of any one of claims 23-25, whereinthe buffered solution has a pH of about 7.4 to about 9.0.
 27. The methodof any one of claims 23-26, wherein the buffered solution comprises45-55% (w/w) ethylene glycol.
 28. The method of any one of the previousclaims, wherein each sugar solution comprises a disaccharide ortrisaccharide.
 29. The method of claim 28, wherein the disaccharidecomprises sucrose, maltose, lactose, and combinations thereof.
 30. Themethod of any one of the previous claims, wherein each sugar solutioncomprises sucrose.
 31. The method of any one of the previous claims,wherein each of the sugar solutions further comprises TrisHCl and NaCl.32. The method of claim 31, wherein the TrisHCl is at a concentration ofabout 20 to about 50 mM and the NaCl is at a concentration of about 150mM to about 500 mM.
 33. The method of claim 31 or claim 32, wherein thebuffered solution has a pH of about 7.4 to about 8.5.
 34. The method ofany one of the previous claims, comprising operating the ultracentrifugeat a first rotational speed of less than 10,000 rpm for less than 60minutes, and at a second rotational speed within the range of about30,000 to about 40,000 rpm for at least 4 hours.
 35. The method of anyone of the previous claims, comprising operating the ultracentrifuge ata first rotational speed of less than 10,000 rpm for less than 60minutes, and at a second rotational speed within the range of about30,000 to about 40,000 rpm for at least 12 hours.
 36. The method ofclaim 34 or claim 35, wherein the first rotational speed is about 3,000rpm to about 6,000 rpm, optionally, about 4,000 rpm.
 37. The method ofclaim 34 or claim 36, wherein the second rotational speed is about35,000 rpm and optionally is maintained for about 4 to about 6 hours.38. The method of any one of claims 34-36, wherein the second rotationalspeed is maintained for at least about 16 hours or at least 20 hours.39. The method of any one of claim 34-36 or 38, wherein the secondrotational speed is about 35,000 rpm and optionally is maintained forabout 16 to about 20 hours.
 40. The method of any one of the previousclaims, wherein the concentrated AAV fraction loaded into the zonalrotor comprises at least 1×10¹² AAV capsids per mL.
 41. The method ofany one of the previous claims, comprising harvesting a supernatant froma cell culture comprising HEK293 cells transfected with a triple plasmidsystem.
 42. The method of claim 41, comprising harvesting thesupernatant about 3 to about 5 days after transfection of the HEK293cells or when the cell culture has a cell density of greater than orabout 5×10⁶ cells/mL and has a cell viability of greater than 50%. 43.The method of claim 41 or claim 42, comprising filtering the harvestedsupernatant via depth filtration.
 44. The method of claim 43, comprisingfiltering the harvested supernatant through a filter comprisingcellulose and perlites and having a minimum permeability of about 500L/m².
 45. The method of claim 43, comprising filtering the harvestedsupernatant through a filter with a minimum pore size of about 0.2 μm.46. The method of any one of the previous claims, comprising (i)applying an AAV fraction to an anion exchange (AEX) chromatographycolumn under conditions that allow for the AAV to flow through the AEXchromatography column and (ii) collecting the flow-through comprisingthe AAV.
 47. The method of claim 46, wherein the AAV fraction is appliedto the AEX chromatography column with a loading buffer comprising about100 mM to about 150 mM NaCl, optionally, wherein the pH of the loadingbuffer is about 8 to about
 9. 48. The method of claim 47, wherein theloading buffer comprises about 115 mM to about 130 mM NaCl, optionally,wherein the loading buffer comprises about 120 mM to about 125 mM NaCl.49. The method of any one of the previous claims, comprisingconcentrating an AAV fraction using an ultra/diafiltration system. 50.The method of claim 49, comprising concentrating an AAV fraction usingan ultra/diafiltration system before, after, or before and after a stepcomprising applying an AAV fraction to an anion exchange (AEX)chromatography column under conditions that allow for the AAV to flowthrough the AEX chromatography column.
 51. The method of any one of theprevious claims, wherein host cell proteins are removed.
 52. The methodof claim 51, wherein the host cell proteins are HSP70 and/or LDH. 53.The method of any one of the previous claims, further comprisinginactivating lipid enveloped viruses of an AAV fraction with a solventand/or detergent.
 54. The method of any one of the previous claims,further comprising nanofiltration of an AAV fraction to remove virusesgreater than 35 nm.
 55. The method of any one of the previous claims,further comprising a polish step comprising performing AEXchromatography with a column comprising tentacle gel.
 56. A method ofproducing an AAV product comprising (i) transfecting host cells withthree plasmids, (ii) collecting a supernatant of a cell culturecomprising the transfected host cells, and (iii) carrying out a methodin accordance with any one of claims 1-55.
 57. The method of claim 56,wherein the steps of the method occur in the order as shown in FIG. 1.58. The method of any one of the previous claims, comprising testing anAAV fraction via an AAV-specific ELISA.
 59. The method of claim 58,wherein the method does not include a step of measuring potency viaquantitative PCR.
 60. The method of claim 58 or claim 59, wherein theAAV specific ELISA is a sandwich ELISA specific for AAV.
 61. The methodof any one of the previous claims, wherein the method comprises a firstsugar solution comprising a sugar at a concentration equivalent to asucrose concentration of about 55%, a second sugar solution comprising asugar at a concentration equivalent to a sucrose concentration of about60%, and optionally a third sugar solution comprising a sugar at aconcentration equivalent to a sucrose concentration of about 50%. 62.The method of any one of the previous claims, wherein AAV is AAV1, AAV2,AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10
 63. The method of anyone of the previous claims, wherein AAV is AAV8.
 64. An AAV productproduced by a method according to any one of claims 1-63.
 65. Apharmaceutical composition comprising an AAV product produced by amethod according to any one of claims 1-63.